Influence of Agro-Waste Substrates on the Growth, Nutritional Composition, Phytochemical Profile, Antimicrobial, and Antioxidant Properties of Pleurotus pulmonarius and Pleurotus florida

Influence of Agro-Waste Substrates on the Growth, Nutritional Composition, Phytochemical Profile, Antimicrobial, and Antioxidant Properties of Pleurotus pulmonarius and Pleurotus florida | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Influence of Agro-Waste Substrates on the Growth, Nutritional Composition, Phytochemical Profile, Antimicrobial, and Antioxidant Properties of Pleurotus pulmonarius and Pleurotus florida Mubarak Muhammed Abdulrazaq, Toheeb Taiye Bajepade, Muhammad Abdulrahman, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8055233/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 12 You are reading this latest preprint version Abstract Agro-based wastes provide sustainable and nutrient-rich alternatives to synthetic substrates for mushroom cultivation. This study examined the effects of different substrates on the nutritional composition, phytochemical profile, antimicrobial, and antioxidant properties of Pleurotus pulmonarius and Pleurotus florida . Corn cob, sawdust, and compost waste were used to assess their influence on growth performance and bioactive compound production. Standard analytical methods were employed to determine proximate composition, total phenolics, flavonoids, and antioxidant capacity, while antimicrobial activities were evaluated against selected bacterial and fungal pathogens using aqueous extracts. High-performance liquid chromatography (HPLC) was used to quantify aflatoxin residues. Results revealed that sawdust supported the highest mycelial growth and yield, followed by corn cob, while compost waste produced lower performance. P. florida exhibited superior antioxidant and antibacterial activities compared to P. pulmonarius , with the highest inhibition recorded against Staphylococcus aureus and Escherichia coli . Both species also showed strong antifungal effects against Aspergillus species. Trace concentrations of aflatoxin B₁, B₂, and G₁ detected in the samples suggest possible substrate-related contamination rather than endogenous production. These findings demonstrate that substrate composition significantly affects the nutritional and bioactive properties of Pleurotus species. The use of lignocellulosic agro-wastes such as sawdust and corn cob not only enhances mushroom productivity and bioactivity but also promotes sustainable waste management and functional food development. Pleurotus species agro-waste substrates antioxidant activity antimicrobial properties aflatoxin sustainable cultivation Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 1.0 Introduction Mushrooms have long been valued not only as food but also for their remarkable medicinal properties (Łysakowska et al., 2023 ). As functional foods, they serve a dual role by providing essential nutrients such as proteins, dietary fiber, amino acids, B-complex vitamins, vitamin D, and minerals like potassium, phosphorus, and selenium while also promoting health beyond basic nutrition (Awuchi et al., 2020 ). Rich in bioactive compounds like polysaccharides (notably β-glucans), phenolics, terpenoids, and flavonoids, mushrooms exhibit antioxidant, antimicrobial, anti-inflammatory, and anticancer properties (Kumar et al., 2021 ). These attributes make them valuable in preventive health and disease management, particularly for combating oxidative stress, modulating immunity, and reducing the risk of chronic illnesses such as cancer, diabetes, and cardiovascular diseases (Muscolo et al., 2024 ). Medicinal species like Ganoderma lucidum (Reishi), Lentinula edodes (Shiitake), and Hericium erinaceus (Lion’s Mane) have been extensively studied in traditional Chinese and Ayurvedic medicine. They are now gaining global recognition in evidence-based nutraceutical research (Torres-Gómez and Guevara, 2025 ). Growing mushrooms is essential to biotechnology and sustainable agriculture. Through the use of agricultural waste (e.g., sawdust, rice straw, corn cobs), it transforms low-value substrates into nutritious food while reducing environmental waste and supporting circular economies (Bibi et al., 2023 ). Unlike traditional crops, mushrooms require minimal land, water, and time, making them ideal for resource-limited settings (Zhang et al., 2025 ). Additionally, cultivation offers economic opportunities, particularly for rural communities. From a biotechnological perspective, mushrooms are biofactories for enzymes, bioactive metabolites, and mycoproteins (Lübeck and Lübeck, 2022 ). Their potential for industrial fermentation and bioremediation stems from their capacity to grow under controlled conditions on lignocellulosic substrates. Improvements in strains further increase yield, growing efficiency, and bioactivity, making mushrooms important contributors to the manufacture of functional foods and environmentally friendly medications (Liu et al., 2021 ; Łysakowska et al., 2023 ). Among cultivated species, oyster mushroom ( Pleurotus species), commonly known as ‘Dhingri’ in India, stands out for its nutritional and medicinal benefits. It is rich in vitamin C, B-complex vitamins, and protein (1.6–2.5%), contains essential minerals, and is cholesterol-free, making it easily digestible (Chauhan et al., 2024 ). Its high potassium-to-sodium ratio supports cardiovascular health, while its folic acid content helps combat anemia (Ren et al., 2024 ). Oyster mushrooms also exhibit antitumor, antioxidant, and antihyperglycemic properties, contributing to their global cultivation. Their adaptability to diverse agro-climatic conditions and ability to grow on agricultural waste further enhance their sustainability (Dimopoulou et al., 2025 ). Mushroom cultivation of Pleurotus species holds promise for improving nutrition, health, and sustainability. However, the inadequate optimization of growth substrates remains a major challenge. The composition and quality of cultivation substrates strongly influence mycelial development, productivity, nutrient content, and secondary metabolite synthesis, while unstandardized use of agricultural residues can introduce mycotoxin contamination risks. To address these challenges, this study investigated how different agro-waste substrates, corn cob, sawdust, and compost waste, affect the growth performance, nutritional composition, and bioactive potential of Pleurotus pulmonarius and Pleurotus florida . It further evaluated morphological traits, proximate composition, aflatoxin contamination, and the mushrooms' antioxidant and antimicrobial capacities to ensure both quality and safety. This study was designed to establish safe and sustainable cultivation practices for Pleurotus species using agro-wastes, enhancing nutritional quality while promoting agricultural waste recycling. Overall, this work provides insight into substrate-dependent optimization for enhanced yield and metabolite production, supporting the advancement of Pleurotus cultivation as a reliable source of functional foods and natural therapeutic agents. 2.0 Materials and Methods 2.1 Materials Pleurotus pulmonarius and Pleurotus florida were obtained from a commercial supplier in Lagos State, Nigeria. The corn cobs were sourced from Malete, while sawdust was collected from Sawmill Road in Ilorin, Kwara State, under sterile conditions. Compost waste was also obtained from Malete. Furthermore, all mushroom cultivation procedures were conducted in the microbiology laboratory at Kwara State University, Malete. The cultivation was performed under ambient laboratory conditions without controlled temperature, humidity, or ventilation. 2.2 Cultivation Procedure of Pleurotus Species The cultivation of Pleurotus pulmonarius and Pleurotus florida was carried out using three different substrates: corn cob, sawdust, and compost waste. The corn cob and sawdust substrates were thoroughly washed to remove dirt and debris, then moistened with distilled water to attain optimal moisture content. Compost waste, however, was sterilized directly without prior washing. All substrates were sterilized at 121°C for 15 minutes to eliminate microbial contaminants and subsequently allowed to cool to room temperature. After sterilization and cooling, 1 kg of each substrate was weighed and packed into heat-resistant polypropylene bags. Equal quantities of Pleurotus pulmonarius and Pleurotus florida spawn were inoculated into each substrate layer by layer under aseptic conditions. The bag openings were sealed with sterile cotton wool and covered with paper to prevent contamination during incubation. The inoculated bags containing corn cob, sawdust, and compost waste were incubated in a dark room at 20–25°C for one week to initiate mycelial growth. Relative humidity was maintained between 70% and 85% by regularly spraying water on gunny bags covering the incubation chamber. Once full mycelial colonization and pinhead (primordia) formation were observed, small perforations were made in the bags to allow aeration and facilitate fruiting. During the fruiting phase, the bags were watered twice daily (morning and evening) to maintain adequate moisture, and diffused light was provided to promote proper fruit body development. Contaminated or poorly colonized bags were immediately discarded to prevent the spread of infection. Mature mushrooms were harvested by gently twisting the fruiting bodies from the substrate after six weeks (42 days) for corn cob and sawdust substrates, while those grown on compost waste were harvested after seven weeks (49 days) due to slower mycelial colonization and fruiting development. After harvesting, the bags were returned to the growing chamber for subsequent flushes. This cultivation procedure was adapted from Chai et al. ( 2021 ), Jasinska and Siwulski ( 2021 ), and Mishra et al. ( 2024 ). 2.3 Growth and Yield Data Collection of Pleurotus Species Growth and yield performance of Pleurotus pulmonarius and Pleurotus florida cultivated on the three substrates were evaluated following the methods of Chukwu et al. ( 2022 ) and Bandura et al . (2024). Data collection focused on the number, weight, and morphological dimensions of the fruiting bodies, as well as the time required for developmental stages. The time required for primordial initiation was determined by monitoring randomly selected bags from each substrate daily after incubation. The number of days from the completion of mycelial colonization to the appearance of the first primordia was recorded. The time required for harvest was measured from the day of primordial initiation to the maturity of the fruiting bodies, and mean values were computed. The total number of primordia and effective mature fruiting bodies per bag was counted and averaged across replicates. The fresh weight of individual fruiting bodies was measured using a digital weighing balance, and the average yield per bag was calculated. Morphological parameters such as height, stipe length, and cap diameter were measured in centimeters using a transparent ruler, and average values for each parameter were recorded for comparison among substrates. 2.4 Proximate Composition of Cultivated Mushroom The proximate composition of the cultivated Pleurotus pulmonarius and Pleurotus florida samples grown on three different substrates, corn cob, sawdust, and compost waste, was analyzed to evaluate the nutritional composition of each species across the substrates. The analysis was conducted according to the standard AOAC procedures described by AOAC ( 2019 ), determining key nutritional parameters including moisture content, crude protein, total ash, crude fibre, crude fat, and total carbohydrate. 2.5 Determination of Aflatoxin Compounds in Pleurotus pulmonarius and Pleurotus florida using HPLC-FLD Analysis 2.5.1 Sample Preparation Fresh fruiting bodies of Pleurotus pulmonarius and Pleurotus florida were harvested, cleaned to remove debris, and air-dried at 50°C until a constant weight was reached. The dried mushrooms were pulverized into fine powder using a laboratory mill and stored in amber glass bottles at 4°C until analysis. For extraction, 10 g of powdered sample was weighed into a 250 mL Erlenmeyer flask, and 50 mL of methanol–acetonitrile–water (60:30:10, v/v/v) was added. The mixture was vortexed for 5 min and mechanically shaken for 20 min at room temperature, then centrifuged at 5000 rpm for 10 min. The supernatant was collected for cleanup (Ahmed et al ., 2023). 2.5.2 Immunoaffinity Column Clean-Up To purify the extract and remove matrix interferences, 20 mL of the clarified extract was diluted with 30 mL of phosphate-buffered saline (PBS, 0.1 M, pH 7.4) and filtered through glass microfiber paper. A 25 mL portion of the filtrate (equivalent to 1 g of sample) was passed through an AflaTest® immunoaffinity column (VICAM, USA) at a steady flow rate (~ 1 mL/min) under gravity. After sample loading, the column was washed with 5 mL PBS and 5 mL distilled water, then gently air-dried. The aflatoxins were eluted using 2 mL HPLC-grade methanol into amber vials. The eluate was filtered through a 0.45 µm PTFE syringe filter into HPLC vials without derivatization (Ahmed et al ., 2023). 2.5.3 HPLC-FLD Analysis Quantitative determination of aflatoxins was carried out using a Shimadzu LC-20A HPLC system equipped with a fluorescence detector (FLD). Separation was achieved on a reversed-phase C18 analytical column (150 mm × 4.6 mm, 5 µm) maintained at 30°C. The mobile phase consisted of water–methanol–acetonitrile (60:20:20, v/v/v) under isocratic elution at a flow rate of 1.0 mL/min, and the injection volume was 20 µL. The detector was set at an excitation wavelength of 365 nm and an emission wavelength of 455 nm. Distinct chromatographic peaks corresponding to aflatoxins B₂, B₁, and G₁ were observed at retention times of approximately 1.3, 1.9, and 3.1 minutes, respectively (Ahmed et al ., 2023). 2.5.4 Calibration and Quantification Standard stock solutions of aflatoxins B₁, B₂, G₁, and G₂ (Sigma-Aldrich, USA) were prepared in acetonitrile and diluted to working concentrations (0.2–10 ng/mL). Calibration curves were plotted as peak area versus concentration for each aflatoxin, showing linearity with correlation coefficients (r² >0.999). Sample concentrations were calculated from the standard curves and expressed as µg/kg (ppb) of dry mushroom weight. Peaks were identified by comparing retention times of samples with those of aflatoxin standards analyzed under identical conditions (Ahmed et al ., 2023). 2.5.5 Method Validation The analytical method was validated for selectivity, linearity, precision, and recovery. Blank mushroom powder confirmed to be aflatoxin-free was spiked with standards at 2, 5, and 10 µg/kg levels and analyzed in triplicate. Mean recoveries ranged from 82–108%, with relative standard deviations (RSD) < 10%. Limits of detection (LOD) and quantification (LOQ) corresponded to signal-to-noise ratios of 3:1 and 10:1, respectively. Chromatograms were processed using Shimadzu LabSolutions software, and all results were expressed as mean ± standard deviation of triplicate determinations (Ahmed et al ., 2023). 2.6. Qualitative Phytochemical Screening The qualitative phytochemical screening of Pleurotus pulmonarius and Pleurotus florida samples for alkaloids, flavonoids, terpenoids, saponins, and tannin was carried out using standard methods described by Herawati et al. ( 2021 ). 2.6.1 Alkaloid Test The presence of alkaloids in the extracts of Pleurotus pulmonarius and Pleurotus florida was determined using Dragendorff’s reagent. The reagent was prepared by dissolving 0.5 g of bismuth (III) nitrate in 6 mL of glacial acetic acid and 24 mL of distilled water (Solution I), while Solution II consisted of 12 g of potassium iodide dissolved in 30 mL of distilled water. Equal volumes (1 mL each) of the two solutions were mixed, followed by the addition of 2 mL of acetic acid and 10 mL of distilled water to obtain the working Dragendorff solution. For the test, 5 mL of each mushroom extract was mixed with 2 mL of potassium chloride (KCl) solution and 1 mL of Dragendorff reagent. The formation of an orange or reddish coloration indicated the presence of alkaloids. 2.6.2 Flavonoid Test Flavonoids were qualitatively identified by adding a few drops of 1% sodium hydroxide (NaOH) solution to 1 mL of the mushroom extract. The development of a bright yellow coloration that disappeared upon the addition of dilute hydrochloric acid (1% HCl) confirmed the presence of flavonoids in the extracts of P. pulmonarius and P. florida . 2.6.3 Saponin Test For the detection of saponins, 1 mL of each mushroom extract, dissolved in acetone, was transferred into a test tube, and 10 mL of hot distilled water was added. The solution was allowed to cool and then shaken vigorously for about 10 seconds. The formation of a stable foam layer (1–10 cm in height) that persisted for approximately 10 seconds and remained unchanged after the addition of a drop of 2N hydrochloric acid indicated the presence of saponins. 2.6.4 Tannin Test The test was carried out by putting 10 mL of the mushroom extract solution into a test tube and adding 1% Pb (CH 3 COO) 2 solution. Tannins are present when a yellow precipitate is formed during the reaction. 2.6.5 Triterpenoids Test In this method, 1 mL of each Pleurotus pulmonarius and Pleurotus florida extract dissolved in acetone was placed in a test tube, followed by the addition of 10 drops of anhydrous acetic acid and 2 drops of concentrated sulfuric acid. The mixture was gently shaken and allowed to stand for a few minutes to observe any color change. The development of a red or purple coloration indicated the presence of triterpenoids, confirming that terpenoid compounds were present in the mushroom extracts. 2.7. Quantitative Phytochemical Analysis The quantitative determination of phenolic compounds, flavonoids, terpenoids, and essential oils in Pleurotus pulmonarius and Pleurotus florida was carried out using standard spectrophotometric and hydrodistillation methods as described by Ogidi et al. ( 2021 ) and Salachna et al. ( 2021 ). 2.7.1 Determination of Total Phenolic Content The total phenolic content of Pleurotus pulmonarius and Pleurotus florida extracts was determined using a modified Folin–Ciocalteu method. A 200 µL portion of each mushroom extract was mixed with 800 µL of Folin–Ciocalteu reagent and 2 mL of 7.5% sodium carbonate (Na₂CO₃) solution. The mixture was diluted to a final volume of 7 mL with distilled water and incubated in the dark for 2 hours at room temperature. Absorbance was measured at 765 nm using a spectrophotometer. Gallic acid was used as the standard, and total phenolic content was expressed as milligrams of gallic acid equivalents per milliliter of extract (mg GAE/mL). 2.7.2 Determination of Total Flavonoid Content The total flavonoid content of Pleurotus pulmonarius and Pleurotus florida extracts was determined using the aluminum chloride colorimetric method. The mushroom extract was mixed with distilled water and sodium nitrite (NaNO₂) solution. After 6 minutes, aluminum chloride (AlCl₃) solution was added and allowed to stand for another 6 minutes, followed by the addition of sodium hydroxide (NaOH) solution. Distilled water was then added to make up the final volume, and the mixture was thoroughly mixed and left to stand for 15 minutes. The absorbance was measured at 510 nm using a spectrophotometer. Rutin was used as the standard, and total flavonoid content was calculated from the standard calibration curve and expressed as milligrams of rutin equivalents per gram of extract (mg RE/g). 2.7.3 Determination of Total Terpenoid Content One gram of each Pleurotus pulmonarius and Pleurotus florida extract was macerated in 50 mL of ethanol and filtered. To 2.5 mL of the filtrate, 2.5 mL of 5% aqueous phosphomolybdic acid solution and 2.5 mL of concentrated sulfuric acid (H₂SO₄) were added sequentially and mixed thoroughly. The mixture was allowed to stand for 30 minutes, after which the volume was made up to 12.5 mL with ethanol. The absorbance was then measured at 700 nm, and the total terpenoid content was determined accordingly. 2.7.4 Determination of Essential Oil Content Twenty-five grams of dried samples of Pleurotus pulmonarius and Pleurotus florida were each mixed with 400 mL of distilled water and subjected to hydrodistillation for 3 hours using a Clevenger-type apparatus. The resulting essential oil was separated from the aqueous phase, dried over anhydrous sodium sulfate, filtered, and weighed. The oils were then stored in dark, airtight vials at 4°C until further analysis by GC–MS. The essential oil content was calculated on a dry weight basis and expressed as a percentage (% w/w). 2.8. Antioxidant Activities of Cultivated Pleurotus pulmonarius and Pleurotus florida 2.8.1. Nitric Scavenging Assay The nitric oxide scavenging activity of Pleurotus pulmonarius and Pleurotus florida extracts was determined based on the Griess reaction. Briefly, a reaction mixture (3 mL) containing 10 mM sodium nitroprusside and 1 mL of mushroom extract at varying concentrations (50–100 mg/mL) was prepared in phosphate buffer (pH 7.4) and incubated at 25°C for 2.5 hours. After incubation, 1 mL of sulfanilic acid reagent was added to 0.5 mL of the reaction mixture and allowed to stand for 5 minutes, followed by the addition of 1 mL of 0.1% naphthyl ethylenediamine dihydrochloride. The resulting solution was incubated at room temperature for 30 minutes, and absorbance was measured at 540 nm using a UV–visible spectrophotometer (Bristy et al., 2022 ). Ascorbic acid served as the standard, and nitric oxide scavenging activity was expressed as percentage inhibition using the formula: %NO scavenging= \(\:\:\frac{AC-AS}{AC}\times\:100\) Where: AC absorbance of the negative control and AS absorbance of the sample. 2.8.2. Ferric Scavenging Assay The reducing power of Pleurotus pulmonarius and Pleurotus florida extract was measured as given by Boonsong et al. ( 2016 ); Yusuf-Salihu et al. ( 2025 ). Briefly, 1 ml of the extract of varying concentration (50–100 mg/ml) with ascorbic acid serving as the standard reference, 2.5 ml of phosphate buffer (pH 6.6), and 2.5 ml of potassium ferricyanide (30 mM) were added and incubated at 50°C for 20 min. Then, 2.5 ml of trichloroacetic acid (600 mM) was added to the reaction mixture and centrifuged for 10 min at 3000 rpm. The upper layer of the solution (2.5 ml) was mixed with 2.5 ml of distilled water and 0.5 ml of FeCl 3 (6 mM), and absorbance was measured at 700 nm. Ascorbic acid was used as a standard. The percentage inhibition of ferric reducing power was calculated using the formula: % Reducing Power = \(\:\:\frac{AS-AB}{AC-\text{A}\text{B}}\times\:100\) Where : AS = Absorbance of the sample extract AC = Absorbance of the standard AB = Absorbance of reagent blank (without sample or control) 2.8.3. DPPH (2,2-Diphenyl-1-picrylhydrazyl) Radical Scavenging Assay The antioxidant activity of Pleurotus pulmonarius and Pleurotus florida extracts was evaluated using the DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging assay following Boonsong et al. ( 2016 ) and Yusuf-Salihu et al. ( 2025 ). A 1 mM DPPH solution (3 mL) was mixed with 1 mL of mushroom extract at varying concentrations (50–100 mg/mL) and incubated in the dark for 30 minutes at 27 ± 2°C. Ascorbic acid and methanol served as the positive control and blank, respectively. Absorbance was measured at 517 nm using a BIOBASE BK-D590 spectrophotometer, and the percentage inhibition of DPPH radicals was calculated using the formula: %DPPH scavenging = \(\:\:\frac{AC-AS}{AC}\times\:100\) Where: AC absorbance of the negative control and AS absorbance of the sample. 2.8.4. Hydrogen Peroxide Scavenging Assay The hydrogen peroxide scavenging assay was evaluated using the modified method of Bristy et al. ( 2022 ). A solution of H 2 O 2 (40 mM) was prepared in a phosphate buffer of pH 7.4. The concentration of H 2 O 2 was determined by absorption using a spectrophotometer. 4 ml of different concentrations of 50,60,70,80,90, and 100 mg/ml of Pleurotus pulmonarius and Pleurotus florida , and distilled water was added to 0.60 ml, 40 mM of H 2 O 2 solution. The absorbance of H 2 O 2 at 230 nm was determined after 20 min against a blank solution (phosphate buffer with H 2 O 2 ), and ascorbic acid was used as the standard. The percentage of hydrogen peroxide scavenging by the Pleurotus pulmonarius and Pleurotus florida and standard compounds was calculated as follows: % H 2 O 2 Scavenging = \(\:\frac{AC-AS}{AC}\times\:100\) Where: AC absorbance of the negative control and AS absorbance of the sample. 2.9 Antimicrobial Activities of Cultivated Pleurotus pulmonarius and Pleurotus florida 2.9.1 Antibacterial Activity The antibacterial activity of each mushroom aqueous extract was assessed using the broth culture method (Yusuf-Salihu et al., 2025 ). Each bacterial strain was cultured in Nutrient Broth (NB) at 37°C for 24 hours, and the bacterial suspensions were adjusted to a concentration of 10 6 colony-forming units (cfu) per milliliter, equivalent to a 0.5 McFarland standard. Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus , and Pseudomonas aeruginosa were used to inoculate 9 mL of peptone broth supplemented with 1 mL of Pleurotus pulmonarius and Pleurotus florida crude extracts at gradient concentrations of 50 mg/ml, 60 mg/ml, 70 mg/ml, 80 mg/ml, 90 mg/ml, and 100 mg/ml. The control group received no crude extract, while the positive control received 1 mL of chloramphenicol (30 µg/mL). The cultures were then incubated at 37°C for 24 hours. Subsequently, their optical density readings were measured at 600 nm, and the percentage growth inhibition was determined as follows: % Growth inhibition = \(\:\frac{\text{O}\text{D}\:\text{c}\text{o}\text{n}\text{t}\text{r}\text{o}\text{l}-\text{O}\text{D}\:\text{t}\text{e}\text{s}\text{t}}{\text{O}\text{D}\:\text{c}\text{o}\text{n}\text{t}\text{r}\text{o}\text{l}}\times\:100\) 2.9.2 Assay for Antifungal Activity The antifungal activity of Pleurotus pulmonarius and Pleurotus florida extract was evaluated using the agar well diffusion method, with results expressed as zones of inhibition. Agar plates were inoculated with fungal strains Aspergillus fumigatus , A. flavus , and A. niger , and wells of 6 mm diameter were bored using a sterile cork borer. Each well was filled with 100 µL of the mushroom extract at concentrations of 50, 60, 70, 80, and 90 mg/mL, while sterilized distilled water served as the control. After incubation, the antifungal efficacy was determined by measuring the diameter of the inhibition zones around each well (Yusuf-Salihu et al., 2025 ). 2.10. Statistical Analysis All data are presented as mean ± standard deviation (n = 3). A two-way ANOVA was used to assess the effects of mushroom species and substrate type on growth, yield, nutritional, and phytochemical parameters. A one-way ANOVA was applied to analyze concentration-dependent effects in antimicrobial and antioxidant assays. Tukey's HSD post-hoc test was used for multiple comparisons, with significance defined at p < 0.05. Analyses were performed using IBM SPSS Statistics. 3.0 Results 3.1. Morphological Growth of Pleurotus pulmonarius and Pleurotus florida on Different Substrates The morphological growth characteristics of Pleurotus pulmonarius and Pleurotus florida cultivated on three different substrates sawdust, corn cob, and compost waste. Both species exhibited optimal growth on sawdust and corn cob substrates, which produced the highest total yield and well-developed morphological structures. On sawdust, P. pulmonarius and P. florida recorded mean yields of 181.07 ± 0.82 g and 159.05 ± 1.09 g, respectively, with large pileus diameters, longer stipes, and greater total height. Corn cob also supported vigorous growth, with P. florida attaining a slightly higher total height (9.0 ± 0.84 cm) than P. pulmonarius (8.4 ± 1.10 cm), indicating favorable substrate conditions. In contrast, compost waste resulted in the poorest growth performance for both species, with markedly smaller pilei, shorter stipes, and lower overall height, reflecting its limited nutrient availability and less supportive structure (Table 1 ). Table 1 Morphological growth of Pleurotus pulmonarius and Pleurotus florida on different substrates Substrate Total Yield per Bag (g) Pileus (Cap) Diameter (cm) Stipe Length (cm) Total Height (cm) P. pulmonarius P. florida P. pulmonarius P. florida P. pulmonarius P. florida P. pulmonarius P. florida Sawdust 181.07 ± 0.82 159.05 ± 1.09 5.4 ± 0.85 6.1 ± 0.78 4.2 ± 0.65 4.8 ± 0.73 7.3 ± 1.02 8.0 ± 0.95 Corn cob 158.12 ± 1.47 170.40 ± 1.38 6.7 ± 0.85 6.5 ± 0.92 5.4 ± 0.80 5.1 ± 0.68 8.4 ± 1.10 9.0 ± 0.84 Compost waste 37.57 ± 1.42 39.43 ± 0.85 2.1 ± 0.54 2.4 ± 0.49 1.2 ± 0.45 1.8 ± 0.51 2.7 ± 0.66 2.8 ± 0.61 3.2. Yield Performance of Pleurotus pulmonarius and Pleurotus florida on Different Substrates The yield performance of Pleurotus pulmonarius and Pleurotus florida cultivated on different substrates revealed notable variations in growth and fruiting behavior (Table 2 ). The shortest time required for primordial initiation was recorded on sawdust (19 ± 0.00 days for P. pulmonarius and 20 ± 0.00 days for P. florida ), followed closely by corn cob (20 ± 0.00 and 18 ± 0.00 days, respectively). In contrast, mushrooms grown on compost waste exhibited the longest initiation period of 30 ± 0.00 days for both species. Similarly, the time to harvest was consistent across sawdust and corn cob substrates (42 ± 0.00 days) but extended to 49 ± 0.00 days on compost waste, reflecting slower mycelial colonization and fruiting. The number of total primordia and effective fruiting bodies followed a similar trend, with sawdust supporting the highest formation (24 ± 1.00 and 21.67 ± 1.53 primordia; 22 ± 2.00 and 19.67 ± 1.53 fruiting bodies for P. pulmonarius and P. florida , respectively). Corn cob showed moderate productivity, while compost waste produced the lowest number of primordia (10.50 ± 0.50 and 6.67 ± 1.15) and effective fruiting bodies (6.33 ± 1.73 and 6.67 ± 1.15) for the two species (Table 2 ). Table 2 Yield Result of Fruiting Bodies of Pleurotus pulmonarius and Pleurotus florida on Different Substrates Substrate Time required for primordial initiation (day) Time required for harvest (days) Number of total primordial Number of total effective fruiting bodies Pleurotus pulmonarius Pleurotus florida Pleurotus pulmonarius Pleurotus florida Pleurotus pulmonarius Pleurotus florida Pleurotus pulmonarius Pleurotus florida Corn cob 20 ± 0.00 18.±0.00 42 ± 0.00 42 ± 0.00 20 ± 0.816 13.83 ± 1.52 13 ± 2.65 7.33 ± 1.09 Sawdust 19 ± 0.00 20 ± 0.00 42 ± 0.00 42 ± 0.00 24 ± 1.00 21.67 ± 1.53 22 ± 2.0 19.67 ± 1.53 Compost 30 ± 0.00 30 ± 0.00 49 ± 0.00 49 ± 0.00 10.50 ± 0.50 6.67 ± 1.15 6.33 ± 1.73 6.67 ± 1.15 3.3. Proximate Composition of Pleurotus pulmonarius and Pleurotus florida The proximate composition of Pleurotus pulmonarius and Pleurotus florida was significantly affected by both substrate type and species, with a notable interaction between these factors (Table 3 ). Sawdust proved to be the most effective substrate for nutrient accumulation, supporting the highest levels of crude protein and ash. Pleurotus pulmonarius generally demonstrated a greater propensity for accumulating protein and ash than P. florida when grown on sawdust and corn cob. Conversely, cultivation on compost waste resulted in a significantly diminished nutritional profile for both species, highlighting its inadequacy as a sole substrate. Furthermore, a strong species effect was evident in carbohydrate content, with P. florida exhibiting a consistently and significantly higher concentration than P. pulmonarius across all substrates Table 3 Proximate Composition of Pleurotus pulmonarius and Pleurotus florida on Corn Cob, Sawdust, and Compost Waste Substrate Mushroom Species Moisture Content (%) Crude Protein (%) Total Ash (%) Crude Fibre (%) Crude Fat (%) Carbohydrate (%) Corn cob P. florida 85.56 ± 0.06 3.06 ± 0.00 0.98 ± 0.04 1.51 ± 0.02 2.53 ± 0.02 6.35 ± 0.14 P. pulmonarius 88.66 ± 0.01 3.14 ± 0.02 1.50 ± 0.02 1.72 ± 0.02 2.55 ± 0.03 2.45 ± 0.03 Sawdust P. florida 84.42 ± 0.05 3.22 ± 0.02 1.12 ± 0.03 1.63 ± 0.02 2.61 ± 0.03 6.08 ± 0.12 P. pulmonarius 87.12 ± 0.02 3.31 ± 0.02 1.61 ± 0.02 1.84 ± 0.02 2.62 ± 0.03 2.23 ± 0.03 Compost waste P. florida 82.85 ± 0.07 2.48 ± 0.02 0.83 ± 0.03 1.24 ± 0.02 2.02 ± 0.03 4.62 ± 0.10 P. pulmonarius 85.90 ± 0.03 2.68 ± 0.02 1.18 ± 0.02 1.36 ± 0.02 2.12 ± 0.02 1.54 ± 0.03 3.4 Mycotoxin Analysis 3.4.1 Quantitative Analysis of Aflatoxin Compounds in Pleurotus pulmonarius Sample The analysis of the Pleurotus pulmonarius sample revealed the presence of several aflatoxin compounds, as shown in the chromatogram (Fig. 1 ) and summarized in Table 4 . Aflatoxin B₁ was detected at the highest concentration of 6.7152 ppb, followed by Aflatoxin B₂ (1.2025 ppb) and Aflatoxin G₁ (0.9588 ppb). Additional minor peaks were detected, likely representing residual matrix components or degradation products. The concentrations of all detected aflatoxins were below the maximum regulatory limits for food safety. Table 4 Quantitative Analysis of Aflatoxin Compounds in Pleurotus pulmonarius Sample Peak No. Peak ID Ret Time (min) Height Area Concentration (ppb) 1 Solvent front 0.907 78.886 1084.386 - 2 Aflatoxin B2 1.332 1036.629 7272.236 1.2025 3 Unidentified 1.640 56.971 203.329 0.0255 4 Aflatoxin B1 1.948 74.679 532.400 6.7152 5 Unidentified 2.148 97.045 961.400 0.5182 6 Aflatoxin G1 3.157 133.190 1504.000 0.9588 7 Unidentified 4.823 44.421 225.800 0.1162 Note: ppb = µg/kg dry weight. The solvent front is not a compound and is not quantified. 3.4.2 Quantitative Analysis of Aflatoxin Compounds in Pleurotus florida Sample The HPLC analysis of the Pleurotus florida sample confirmed the presence of aflatoxins, as detailed in Table 5 and shown in the chromatogram (Fig. 2 ). The sample contained Aflatoxin B₁ at 4.1774 ppb, Aflatoxin G₁ at 2.7558 ppb, and Aflatoxin B₂ at 0.4163 ppb. Similar to the P. pulmonarius sample, multiple unidentified peaks were present. The total aflatoxin load was lower in P. florida compared to P. pulmonarius .. Table 5 Quantitative Analysis of Aflatoxin Compounds in Pleurotus florid Sample Peak No. Peak ID Ret Time (min) Height Area Concentration (ppb) 1 Solvent front 0.132 35.884 392.150 - 2 Aflatoxin B2 1.115 55.286 293.400 0.4163 3 Aflatoxin B1 1.365 450.000 3545.800 4.1774 4 Unidentified 1.498 460.000 2101.300 1.6984 5 Aflatoxin G1 2.107 118.059 2989.400 2.7558 6 Unidentified 2.448 112.342 1234.000 0.8062 7 Unidentified 3.390 103.882 485.450 0.4577 8 Unidentified 3.573 59.240 791.650 0.5910 9 Unidentified 4.865 73.375 750.700 0.4956 3.5 Qualitative Screening of Phytochemical Compounds in Pleurotus pulmonarius and Pleurotus florida Cultivated on Different Substrates The qualitative screening of anti-nutrients in Pleurotus pulmonarius and Pleurotus florida cultivated on sawdust, corn cob, and compost substrates revealed distinct variations in the presence of alkaloids, flavonoids, terpenoids, saponins, and tannin. Overall, both mushroom species exhibited moderate to high levels of alkaloids and flavonoids, with P. florida grown on sawdust showing the highest flavonoid (+++) and tannin (+++) contents, while P. Pulmonarius on sawdust recorded the highest alkaloid (+++) and saponin (++) concentrations. Samples grown on corn cob and compost substrates displayed relatively lower but consistent levels of these bioactive compounds, suggesting that the substrate type significantly influences the qualitative distribution of anti-nutritional components in both mushroom species. Table 6 Qualitative Phytochemical Screening of Pleurotus pulmonarius and Pleurotus florida cultivated on different substrates Substrate Mushroom species Alkaloids Flavonoids Terpenoids Saponins Tannin Sawdust P. florida ++ +++ + + +++ P. pulmonarius +++ ++ + ++ + Corn cob P. florida ++ +++ ++ + ++ P. pulmonarius ++ ++ + ++ +++ Compost P. florida + ++ + + ++ P. pulmonarius + + + ++ + Where (+) = low presence, (++) = moderate presence, (+++) = high presence of respective phytochemicals. 3.6 Quantitative Composition of Pleurotus pulmonarius and Pleurotus florida Cultivated on Different Substrates The quantitative analysis of Pleurotus pulmonarius and Pleurotus florida cultivated on sawdust, corn cob, and compost substrates revealed notable variations in the concentrations of phenolic compounds, flavonoids, terpenoids, and essential oils. P. florida cultivated on sawdust recorded the highest phenolic (622.94 mg/kg) and flavonoid (27.01 mg/kg) contents, while P. pulmonarius on the same substrate showed slightly lower values. Samples grown on corn cob exhibited relatively high concentrations of bioactive compounds, with P. florida showing the highest flavonoid (30.00 mg/kg) and essential oil (16.50 mg/100 g) levels among all. Conversely, mushrooms grown on compost recorded the lowest phenolic and flavonoid contents, indicating that the type of substrate significantly affects the accumulation of secondary metabolites and essential oils in both Pleurotus species. Table 7 Quantitative phytochemical composition of Pleurotus pulmonarius and Pleurotus florida cultivated on different substrates Substrate Mushroom species Phenolic compounds (mg/kg) Flavonoids (mg/kg) Terpenoids (mg/kg) Essential oils (mg/100 g) Sawdust P. florida 622.94 ± 0.26 27.01 ± 0.04 1.66 ± 0.40 17.02 ± 0.08 P. pulmonarius 601.00 ± 0.55 25.01 ± 0.08 1.84 ± 0.60 15.02 ± 0.05 Corn cob P. florida 600.00 ± 0.20 30.00 ± 0.05 2.00 ± 0.35 16.50 ± 0.10 P. pulmonarius 510.00 ± 0.25 25.00 ± 0.04 1.80 ± 0.30 17.00 ± 0.12 Compost P. florida 550.00 ± 0.30 20.00 ± 0.03 1.60 ± 0.25 15.50 ± 0.15 P. pulmonarius 539.00 ± 0.35 18.00 ± 0.02 1.54 ± 0.28 15.00 ± 0.18 3.7 Antioxidant Activities 3.7.1 Ferric Reducing Activity of Pleurotus florida and Pleurotus pulmonarius The ferric reducing antioxidant power (FRAP) of Pleurotus pulmonarius and Pleurotus florida increased progressively with concentration from 50 to 100 mg/mL, showing a dose-dependent response compared to the standard (Fig. 3 ). At lower concentrations, both species exhibited moderate reducing ability, which intensified at higher concentrations. P. florida consistently showed higher ferric reducing activity than P. pulmonarius , with maximum values of 85% and 78% respectively at 90 mg/mL, while the standard reached 91%. 3.7.2. Nitric Oxide Assay Pleurotus florida and Pleurotus pulmonarius The nitric oxide (NO) scavenging activity of Pleurotus pulmonarius and Pleurotus florida increased progressively with concentration from 50 to 100 mg/mL, indicating a clear dose-dependent antioxidant response (Fig. 4 ). Both species demonstrated appreciable nitric oxide inhibition comparable to the standard, though P. florida consistently exhibited slightly higher activity across all concentrations. At 100 mg/mL, P. florida recorded 84% inhibition, while P. pulmonarius achieved 81%, compared to 92% for the standard. 3.7.3 DPPH Radical Scavenging Activity of Pleurotus pulmonarius and Pleurotus florida The DPPH radical scavenging activity of Pleurotus pulmonarius and Pleurotus florida increased progressively with concentration from 50 to 100 mg/mL, indicating a clear dose-dependent antioxidant response (Fig. 5 ). Both species showed appreciable DPPH scavenging ability comparable to the standard, with P. florida consistently exhibiting slightly higher activity at all concentrations. At 100 mg/mL, P. florida recorded 89% inhibition, while P. pulmonarius achieved 84%, compared to 95% for the standard. 3.7.4 Hydrogen Peroxide Scavenging Activity of Pleurotus pulmonarius and Pleurotus florida The hydrogen peroxide (H₂O₂) scavenging activity of Pleurotus pulmonarius and Pleurotus florida increased progressively with concentration from 50 to 100 mg/mL, showing a clear dose-dependent antioxidant response (Fig. 6 ). Both species exhibited considerable hydrogen peroxide scavenging ability comparable to the standard, with P. florida consistently displaying slightly higher activity across all concentrations. At 100 mg/mL, P. florida recorded 84% inhibition, while P. pulmonarius achieved 80%, compared to 92% for the standard. 3.8. Antimicrobial Activity of Pleurotus Extracts 3.8.1 Antibacterial Activity of Pleurotus Extracts As shown in Fig. 7 and Fig. 8 , the antibacterial activity of Pleurotus pulmonarius and Pleurotus florida extracts increased progressively with higher extract concentrations, demonstrating a dose-dependent inhibitory effect against all tested bacterial isolates. In P. pulmonarius extract (Fig. 7 ), Staphylococcus aureus exhibited the highest inhibition (78.69% at 100 mg/mL), followed by Escherichia coli (70.32%), Pseudomonas aeruginosa (68.76%), and Klebsiella pneumoniae (65.08%). Similarly, P. florida extract (Fig. 8 ) showed maximum inhibition against S. aureus (86.2%) and E. coli (85.3%) at 100 mg/mL, while K. pneumoniae and P. aeruginosa displayed comparatively lower inhibition values. Both extracts exhibited strong antibacterial efficacy when compared with the positive control and no inhibition with the negative control. 3.8.2 Antifungal Activity of Pleurotus florida Extract Against Fungal Strains The aqueous extracts of both Pleurotus pulmonarius and Pleurotus florida exhibited notable, concentration-dependent antifungal activity against Aspergillus fumigatus , A. flavus , and A. niger , as shown in Tables 8 and 9 . For both mushroom species, the zones of inhibition increased progressively with higher extract concentrations, confirming the presence of potent antifungal bioactive compounds. This inhibitory effect was visually evident in the plate assays, where distinct, clear zones surrounded the wells containing the extracts of P. pulmonarius (Fig. 9 ) and P. florida (Fig. 10 ). Among the tested fungi, A. fumigatus showed the highest susceptibility to both extracts. The extract from P. pulmonarius exhibited its strongest inhibition (33 mm) against A. fumigatus at 90 mg/mL, followed by A. niger (30 mm) and A. flavus (20 mm), as presented in Table 8 . Similarly, P. florida extract demonstrated superior antifungal activity, maintaining the largest inhibition zone of 35 mm against A. fumigatus across all concentrations tested, while A. niger and A. flavus showed inhibition zones of 31 mm and 18 mm, respectively, at 90 mg/mL (Table 9 ). Overall, P. florida extract displayed a slightly higher antifungal potency than P. pulmonarius , particularly against A. fumigatus and A. niger , indicating a stronger inhibitory potential. . Table 8 Table Showing Antifungal Activity on Fungal Strains using Pleurotus pulmonarius extract Test organism Zone of Inhibition (mm) 50 mg/ml 60 mg/ml 70 mg/ml 80 mg/ml 90 mg/ml A.fumigatus 20 21 25 31 33 A.flavus 15 17 17 21.5 20 A.niger 22 25 27 27 30 Table 9 Table Showing Antifungal Activity on Fungal Strains Using Pleurotus florida extract Test organism Zone of Inhibition (mm) 50 mg/ml 60 mg/ml 70 mg/ml 80 mg/ml 90 mg/ml A.fumigatus 33 35 35 35 35 A.flavus 12 15 16 17 18 A.niger 10.50 24 29 25 31 Discussions The superior morphological development of Pleurotus pulmonarius and P. florida on sawdust and corn cob substrates reflects the influence of substrate composition on mushroom growth and yield. The high productivity and well-formed fruiting bodies obtained from these substrates can be attributed to their favorable physical and chemical properties particularly their porous structure, balanced carbon-to-nitrogen ratio, and high lignocellulosic content which enhance aeration, moisture retention, enzymatic activity, and mycelial colonization. The fibrous texture of sawdust and corn cob provides an ideal matrix for mycelial penetration and efficient degradation of organic material, supporting the formation of larger pilei, longer stipes, and taller fruiting bodies as observed in Table 1 . In contrast, compost waste exhibited poor performance, likely due to its compact structure, reduced aeration, and nutrient imbalance, which limited mycelial spread and morphological differentiation. These findings align with the observations of Raman et al. ( 2021 ), who reported improved yield and fruit body morphology of Pleurotus species grown on corn- and wood-based lignocellulosic substrates. Similarly, Diamantopoulou et al. ( 2023 ) and Han et al. ( 2024 ) emphasized that the physicochemical composition of substrates especially the quality of lignocellulosic material and carbon-to-nitrogen balance plays a decisive role in determining the growth rate and morphological traits of cultivated mushrooms. Overall, the results of this study show the critical role of substrate selection in optimizing mushroom cultivation, with corn cob and sawdust identified as the most effective substrates for achieving superior morphological and yield characteristics in Pleurotus species. The observed variations in the yield performance of Pleurotus pulmonarius and P. florida across the three substrates reflect the combined effects of nutrient composition, substrate texture, and aeration capacity on fungal colonization and fruiting efficiency. The superior yield obtained on sawdust and corn cob suggests that these substrates provided optimal physicochemical conditions for mycelial development and primordia formation. Sawdust, with its fine and fibrous texture, offers a stable and moisture-retentive environment that supports dense mycelial networks and abundant primordia, leading to higher numbers of effective fruiting bodies. Conversely, the porous structure and balanced carbon-to-nitrogen ratio of corn cob likely facilitated rapid nutrient diffusion and aeration, which favored faster primordial initiation and fruit body expansion despite slightly lower total yields than sawdust. The delayed initiation and reduced yield observed on compost waste can be attributed to its heterogeneous composition, poor structural integrity, and possible microbial competition, which collectively impede mycelial penetration and nutrient uptake. These findings align with the observations of Castorina et al. ( 2023 ), who reported enhanced fruiting and shorter initiation times for Pleurotus species cultivated on maize cob due to its superior porosity and nutrient balance. Similarly, Argaw et al. ( 2023 ) noted that sawdust substrates promote larger and thicker pilei across successive flushes, highlighting their structural stability and sustained nutrient availability, even when overall yield is moderately lower than on leaf-based substrates. The comparatively poor performance of compost waste contrasts with the results of Sultana et al. ( 2021 ), who demonstrated that yield improvements in compost-based systems depend on organic enrichment with additives such as poultry manure or oil cakes. Overall, the present findings emphasize the importance of substrate-specific optimization strategies in Pleurotus cultivation, confirming that sawdust and corn cob provide superior growth environments that enhance both yield and morphological quality, whereas compost waste remains the least effective substrate for productive mushroom cultivation. The detection of aflatoxin compounds in Pleurotus pulmonarius and Pleurotus florida indicates possible uptake or residual contamination from the substrates used during cultivation, as aflatoxins are secondary metabolites typically produced by Aspergillus species that may persist in agricultural residues even after sterilization. The differential aflatoxin profiles observed between the two species, with each accumulating specific aflatoxin analogs at varying levels, suggest species-specific variations in the uptake, bioaccumulation, or metabolic detoxification mechanisms. This finding is supported by Hainghumbi ( 2020 ), who demonstrated that mycotoxin contamination in edible mushrooms was directly influenced by the natural substrates on which they grew. Importantly, the concentrations of all detected aflatoxins in both species remained below the maximum regulatory limits for food safety, indicating that the mushrooms are safe for consumption. Recent studies by Arimboor et al. (2024) and Zapaśnik et al. ( 2025 ) also suggest that Pleurotus species possess intrinsic ligninolytic enzymes, which may enable the partial degradation of aflatoxins through oxidative and hydrolytic pathways, highlighting their potential for natural mycotoxin detoxification. Overall, the results underscore that while trace aflatoxin residues can occur in cultivated mushrooms, they are likely substrate-derived, and careful substrate selection and proper sterilization remain essential for ensuring food safety. The variations in the qualitative and quantitative composition of anti-nutritional and bioactive compounds in Pleurotus pulmonarius and Pleurotus florida cultivated on different substrates can be attributed to differences in substrate composition, nutrient availability, and metabolic adaptability of each mushroom species. Sawdust supported the highest accumulation of alkaloids, flavonoids, and phenolic compounds, likely due to its richness in lignin and cellulose, which induces oxidative enzyme activity and stimulates secondary metabolite synthesis. In contrast, corn cobs, with their balanced carbon-to-nitrogen ratio and moderate lignocellulosic content, promote relatively high levels of flavonoids and essential oils, reflecting their efficient nutrient exchange and aeration properties that favor the biosynthesis of antioxidant compounds. Compost waste, however, exhibited lower concentrations of these metabolites, possibly because of nutrient degradation and lower substrate stability after decomposition, which limits the metabolic pathways responsible for bioactive compound production. These observations align with the findings of Eze et al. ( 2024 ), who reported that variations in substrate composition, such as maize kernel, dry banana leaf, and empty palm fruit bunch, significantly affect the yield, nutritional profile, and phytochemical accumulation in Pleurotus pulmonarius . Likewise, Baptista et al. ( 2023 ) demonstrated that the biochemical composition of Lentinula edodes spent mushroom substrate (SMS) and the extraction conditions markedly influence the concentration of phenolic compounds, antioxidant capacity, and antibacterial activity. Similarly, Verduzco-Oliva and Gutierrez-Uribe et al. (2020) noted who demonstrated that during solid-state fermentation of plant-based lignocellulosic residues, fungi secrete lignocellulolytic enzymes such as ligninases, cellulases, and hemicellulases that break down the fiber structure, thereby releasing phenolic compounds and stimulating the biosynthesis of additional bioactive metabolites. This indicates that lignocellulosic substrates like sawdust can enhance phenolic and flavonoid synthesis through their structural complexity and availability of lignin-derived precursors. Overall, this study reinforces that the type and quality of substrate not only determine mushroom yield but also significantly affect their phytochemical richness, with sawdust and corn cob emerging as superior substrates for producing nutritionally and medicinally valuable Pleurotus mushrooms. The antioxidant activities exhibited by Pleurotus florida and Pleurotus pulmonarius are attributed to their rich composition of phenolic compounds, flavonoids, and essential oils, which act as potent free radical scavengers and reducing agents. The consistently higher antioxidant performance of P. florida across the ferric reducing, nitric oxide, DPPH, and hydrogen peroxide assays suggests that this species possesses a more efficient redox system, possibly due to higher concentrations of bioactive metabolites derived from substrate interactions. The use of sawdust and corn cob as substrates likely contributed to this enhanced antioxidant capacity, as both materials are rich in lignin and cellulose, which stimulate the production of secondary metabolites involved in oxidative defense. Compost waste, in contrast, may provide fewer biochemical precursors for phenolic synthesis, resulting in comparatively weaker antioxidant responses. Similarly, Diamantopoulou et al. ( 2023 ) demonstrated that the antioxidant activity of Pleurotus mushrooms strongly correlates with substrate composition and phenolic content, while Rusu et al . (2022 ) found that supplementing cultivation substrates with brewery-spent grains significantly enhanced protein, flavonoid, and phenolic levels, leading to improved antioxidant activity. Similarly, Kumla et al. ( 2020 ) reported that mushrooms cultivated on lignocellulosic agro-industrial wastes exhibit enhanced antioxidant activity due to increased production of lignocellulolytic enzymes that stimulate the synthesis of phenolic and flavonoid antioxidants. Overall, this study indicates that substrate type and bioactive compound composition significantly influence antioxidant potential, with P. florida particularly when cultivated on nutrient-balanced substrates like corn cob and sawdust, showing greater capacity to neutralize free radicals and mitigate oxidative stress. The antimicrobial efficacy demonstrated by the Pleurotus extracts in this study shows the growing recognition of edible mushrooms as reservoirs of bioactive compounds with therapeutic potential. Rather than being an incidental property, this antimicrobial activity likely reflects a complex interplay between species-specific metabolic pathways and substrate-induced biochemical modulation. The superior bioactivity observed in Pleurotus florida compared with P. pulmonarius suggests a differential expression of secondary metabolite biosynthetic enzymes, such as laccases, peroxidases, and phenol oxidases, which are known to be influenced by environmental and nutritional factors. The role of lignocellulosic substrates, particularly sawdust and corn cob, appears especially important, as they contain lignin derivatives and cellulose fragments that act as metabolic inducers for phenolic and terpenoid biosynthesis. These compounds are well-documented for their ability to disrupt microbial cell wall integrity, alter membrane permeability, and interfere with essential enzymatic processes. From an ecological and biochemical standpoint, substrate composition shapes not only the growth dynamics of Pleurotus species but also the qualitative and quantitative distribution of their secondary metabolites. Lignin-rich materials tend to favor oxidative enzyme activity, which enhances the formation of complex phenolic molecules with antimicrobial and antioxidant properties. In contrast, substrates with lower aromatic content, such as compost waste, may not sufficiently stimulate these enzymatic pathways, leading to a comparatively weaker bioactive profile. These findings support the hypothesis proposed by Diamantopoulou et al. ( 2023 ) and Kumla et al. ( 2020 ), who emphasized the substrate-dependent modulation of bioactive compound synthesis in basidiomycetes. The notable antimicrobial spectrum of Pleurotus species, spanning both Gram-positive and Gram-negative bacteria as well as common fungal pathogens, highlights their pharmacological versatility. This broad activity range suggests that the extracts act through multiple molecular mechanisms rather than a single inhibitory pathway. Polyphenolic compounds, for instance, have been shown to chelate metal ions, scavenge reactive oxygen species, and form complexes with microbial proteins, thereby impeding microbial metabolism and replication. The coexistence of flavonoids, alkaloids, and terpenoids may further enhance this effect through synergistic interactions that potentiate cellular disruption and biofilm inhibition. Moreover, the variations observed between the two Pleurotus species could also stem from inherent genetic and enzymatic differences. P. florida is known for its rapid mycelial colonization and superior enzymatic adaptability, which may enhance its ability to transform complex substrates into high-value secondary metabolites. This aligns with Illuri et al. ( 2022 ), who attributed the strong antimicrobial capacity of P. florida to its rich profile of flavonoids, anthraquinones, and terpenoids. Such species-specific attributes underline the importance of genetic and physiological factors in determining the pharmacological potential of cultivated mushrooms. Collectively, these findings reinforce the view that substrate optimization and species selection are central to maximizing the antimicrobial efficacy of Pleurotus mushrooms. By strategically manipulating growth media rich in lignocellulosic components, it may be possible to enhance the yield of bioactive compounds with pharmaceutical relevance. The present results, therefore, contribute to a growing body of evidence supporting the integration of mushroom-based extracts into natural antimicrobial formulations, offering sustainable alternatives to synthetic agents in the ongoing fight against multidrug-resistant pathogens. Conclusion This study demonstrated that Pleurotus pulmonarius and Pleurotus florida's nutritional quality, phytochemical content, and antibacterial capability are significantly influenced by their substrate composition. Because of its advantageous lignocellulosic composition and aeration qualities, sawdust promoted superior mycelial development and increased antioxidant and antibacterial activity among the studied substrates. The potential of both Pleurotus species as natural sources of bioactive chemicals was confirmed by their significant antibacterial activity against both Gram-positive and Gram-negative bacteria as well as their mild antifungal activities. Only trace amounts of aflatoxins are present, which suggests substrate-related contamination rather than the mushrooms' natural ability to produce poisons. Overall, the results demonstrate the usefulness of agricultural wastes such sawdust and corn cob as effective, secure, and long-lasting substrates for the production of mushrooms. Overall, the findings highlight the value of agro-based wastes such as sawdust and corn cob as efficient, safe, and sustainable substrates for mushroom cultivation. These results emphasize the dual benefits of improved food security and environmental protection through the conversion of agricultural residues into nutritionally rich and bioactive functional foods. Recommendation Future research should focus on optimizing substrate formulations to maximize bioactive compound synthesis while minimizing mycotoxin contamination. The inclusion of nutrient supplements such as agricultural by-products or trace minerals may further enhance yield, antioxidant potency, and overall nutritional quality. Strict sterilization and mycotoxin monitoring protocols are essential to ensure food safety and product consistency. Comparative metabolomic and enzymatic studies are recommended to clarify the biochemical pathways involved in aflatoxin degradation and antioxidant mechanisms in Pleurotus species. Furthermore, scaling up cultivation using locally available agro-wastes such as corn cob, rice straw, and sawdust can promote sustainable mushroom farming, particularly in rural communities. Strengthening collaborations among researchers, policymakers, and farmers will be vital for integrating mushroom cultivation into circular bioeconomy initiatives, advancing environmental conservation, economic resilience, and functional food development. Declarations Competing Interests The authors have no relevant financial or non-financial interests to disclose. Ethics Approval This study did not involve human participants or vertebrate animals; therefore, ethics approval was not required. All experimental procedures complied with institutional and national standards for laboratory biosafety and good scientific practice. Consent to Participate Not applicable; this study did not involve human participants. Consent to Publish Not applicable; this study does not include any person’s data, images, or other identifying materials. Funding The authors declare that no funds, grants, or other financial support were received during the preparation and execution of this research work. Author Contribution All authors contributed to the study conception and design. Material preparation, data collection, and analysis were performed by Mubarak Muhammed Abdulrazaq, Bajepade Toheeb Taiye, Abdulrahman Muhammad, and Adeyemi Joshua Segun. Mubarak Muhammed Abdulrazaq wrote the first draft of the manuscript, and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript. Acknowledgement The authors sincerely acknowledge the Department of Microbiology, Kwara State University, Malete, Nigeria, for providing the laboratory facilities, technical support, and an enabling research environment for this study. Special appreciation is extended to the technical staff of the Microbiology Laboratory for their assistance during the mushroom cultivation and analytical procedures.The authors also appreciate the valuable guidance and constructive suggestions received from colleagues and academic mentors during the design, analysis, and interpretation of data. Data Availability The datasets generated and/or analyzed during the current study are available from the corresponding author upon reasonable request. 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Supplementary Files Screenshot20251119200402.png Graphical Abstract Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 19 Jan, 2026 Reviews received at journal 22 Dec, 2025 Reviews received at journal 15 Dec, 2025 Reviewers agreed at journal 10 Dec, 2025 Reviewers agreed at journal 08 Dec, 2025 Reviewers agreed at journal 08 Dec, 2025 Reviewers agreed at journal 12 Nov, 2025 Reviewers agreed at journal 11 Nov, 2025 Reviewers invited by journal 10 Nov, 2025 Editor assigned by journal 09 Nov, 2025 Submission checks completed at journal 08 Nov, 2025 First submitted to journal 07 Nov, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8055233","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":544030183,"identity":"af0dcdbd-bda2-4e05-8f9d-814e53ac04be","order_by":0,"name":"Mubarak Muhammed 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1","display":"","copyAsset":false,"role":"figure","size":46913,"visible":true,"origin":"","legend":"\u003cp\u003eHPLC Chromatogram of Aflatoxin Compounds in \u003cem\u003ePleurotus pulmonarius \u003c/em\u003eSample\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8055233/v1/e52dc3e35ed46ce78ba1d2fc.png"},{"id":96365816,"identity":"30ff66eb-8a87-4477-94d1-1a96eaaa87bf","added_by":"auto","created_at":"2025-11-20 10:10:49","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":44947,"visible":true,"origin":"","legend":"\u003cp\u003eHPLC Chromatogram of Aflatoxin Compounds in \u003cem\u003ePleurotus florida\u003c/em\u003e Sample\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8055233/v1/ba2f61e464091f8b7a7a90cd.png"},{"id":96304364,"identity":"0172c6eb-4750-4adf-97f8-f96153ae4adf","added_by":"auto","created_at":"2025-11-19 15:02:53","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":53919,"visible":true,"origin":"","legend":"\u003cp\u003eFerric reducing antioxidant power (FRAP) of aqueous extracts from \u003cem\u003ePleurotus pulmonarius \u003c/em\u003eand \u003cem\u003ePleurotus florida\u003c/em\u003e compared to a standard antioxidant\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8055233/v1/e997b6cf518cba0980d9b3a4.png"},{"id":96304376,"identity":"5bb54999-9f45-4556-88ab-86557c04cc39","added_by":"auto","created_at":"2025-11-19 15:02:53","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":59930,"visible":true,"origin":"","legend":"\u003cp\u003eNitric oxide (NO) scavenging activity of aqueous extracts from \u003cem\u003ePleurotus pulmonarius \u003c/em\u003eand \u003cem\u003ePleurotus florida\u003c/em\u003e compared to a standard 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6","display":"","copyAsset":false,"role":"figure","size":54256,"visible":true,"origin":"","legend":"\u003cp\u003eHydrogen peroxide (H₂O₂) scavenging activity of aqueous extracts from \u003cem\u003ePleurotus pulmonarius \u003c/em\u003eand \u003cem\u003ePleurotus florida\u003c/em\u003e compared to a standard antioxidant\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-8055233/v1/6149f87eac97279abd0d56ce.png"},{"id":96365781,"identity":"3f864363-e05f-4598-bf8a-2123637ffca0","added_by":"auto","created_at":"2025-11-20 10:10:46","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":62399,"visible":true,"origin":"","legend":"\u003cp\u003eAntibacterial activity of \u003cem\u003ePleurotus pulmonarius \u003c/em\u003eextract shown as percentage inhibition against \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e, \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e, \u003cem\u003eEscherichia coli\u003c/em\u003e, and \u003cem\u003eStaphylococcus aureus\u003c/em\u003e across a concentration gradient from 50 to 100 mg/mL, including positive and negative controls\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-8055233/v1/51e3849822195d17fc88d9bb.png"},{"id":96304374,"identity":"3b1ef56d-1616-4b30-ab8e-ff07e14978e0","added_by":"auto","created_at":"2025-11-19 15:02:53","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":62269,"visible":true,"origin":"","legend":"\u003cp\u003eAntibacterial activity of \u003cem\u003ePleurotus florida\u003c/em\u003e\u003cstrong\u003e \u003c/strong\u003eextract shown as percentage inhibition against \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e, \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e, \u003cem\u003eEscherichia coli\u003c/em\u003e, and \u003cem\u003eStaphylococcus aureus\u003c/em\u003e across a concentration gradient from 50 to 100 mg/mL, including positive and negative controls\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-8055233/v1/80c4b6f2bfe68fb663aabff6.png"},{"id":96304377,"identity":"5b0649ee-9318-4940-91b2-41a0977e1fd6","added_by":"auto","created_at":"2025-11-19 15:02:53","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":376763,"visible":true,"origin":"","legend":"\u003cp\u003ePlate assay (A: \u003cem\u003eA. flavus\u003c/em\u003e; B: \u003cem\u003eA. fumigatus\u003c/em\u003e; C: \u003cem\u003eA. niger\u003c/em\u003e) for antifungal activity using \u003cem\u003ePleurotus pulmonarius\u003c/em\u003e\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-8055233/v1/0022973ff7bbca37a81e7c25.png"},{"id":96364746,"identity":"8fe8e7d8-343e-4598-b8bc-ec552407883c","added_by":"auto","created_at":"2025-11-20 10:09:35","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":372767,"visible":true,"origin":"","legend":"\u003cp\u003ePlate assay (A: \u003cem\u003eA. fumigatus\u003c/em\u003e; B: \u003cem\u003eA. flavus;\u003c/em\u003e C: \u003cem\u003eA. niger\u003c/em\u003e) for antifungal activity using \u003cem\u003ePleurotus florida\u003c/em\u003e\u003c/p\u003e","description":"","filename":"10.png","url":"https://assets-eu.researchsquare.com/files/rs-8055233/v1/ce4194758d3e5959f68fe600.png"},{"id":96366763,"identity":"5309b7bb-d171-4e1e-9632-a11b1ac76310","added_by":"auto","created_at":"2025-11-20 10:11:52","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3789120,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8055233/v1/e7d1a8b8-da5d-48b6-a6d4-d778478aecd0.pdf"},{"id":96304363,"identity":"850b164a-284d-454e-8163-cefde6bd68b9","added_by":"auto","created_at":"2025-11-19 15:02:53","extension":"png","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":635900,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGraphical Abstract\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Screenshot20251119200402.png","url":"https://assets-eu.researchsquare.com/files/rs-8055233/v1/bb8812a770d5d68fd450b64e.png"}],"financialInterests":"No competing interests reported.","formattedTitle":"Influence of Agro-Waste Substrates on the Growth, Nutritional Composition, Phytochemical Profile, Antimicrobial, and Antioxidant Properties of Pleurotus pulmonarius and Pleurotus florida","fulltext":[{"header":"1.0 Introduction","content":"\u003cp\u003eMushrooms have long been valued not only as food but also for their remarkable medicinal properties (Łysakowska et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). As functional foods, they serve a dual role by providing essential nutrients such as proteins, dietary fiber, amino acids, B-complex vitamins, vitamin D, and minerals like potassium, phosphorus, and selenium while also promoting health beyond basic nutrition (Awuchi et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Rich in bioactive compounds like polysaccharides (notably β-glucans), phenolics, terpenoids, and flavonoids, mushrooms exhibit antioxidant, antimicrobial, anti-inflammatory, and anticancer properties (Kumar et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). These attributes make them valuable in preventive health and disease management, particularly for combating oxidative stress, modulating immunity, and reducing the risk of chronic illnesses such as cancer, diabetes, and cardiovascular diseases (Muscolo et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Medicinal species like \u003cem\u003eGanoderma lucidum\u003c/em\u003e (Reishi), \u003cem\u003eLentinula edodes\u003c/em\u003e (Shiitake), and \u003cem\u003eHericium erinaceus\u003c/em\u003e (Lion\u0026rsquo;s Mane) have been extensively studied in traditional Chinese and Ayurvedic medicine. They are now gaining global recognition in evidence-based nutraceutical research (Torres-G\u0026oacute;mez and Guevara, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2025\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eGrowing mushrooms is essential to biotechnology and sustainable agriculture. Through the use of agricultural waste (e.g., sawdust, rice straw, corn cobs), it transforms low-value substrates into nutritious food while reducing environmental waste and supporting circular economies (Bibi et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Unlike traditional crops, mushrooms require minimal land, water, and time, making them ideal for resource-limited settings (Zhang et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Additionally, cultivation offers economic opportunities, particularly for rural communities. From a biotechnological perspective, mushrooms are biofactories for enzymes, bioactive metabolites, and mycoproteins (L\u0026uuml;beck and L\u0026uuml;beck, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Their potential for industrial fermentation and bioremediation stems from their capacity to grow under controlled conditions on lignocellulosic substrates. Improvements in strains further increase yield, growing efficiency, and bioactivity, making mushrooms important contributors to the manufacture of functional foods and environmentally friendly medications (Liu et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Łysakowska et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eAmong cultivated species, oyster mushroom (\u003cem\u003ePleurotus\u003c/em\u003e species), commonly known as \u0026lsquo;Dhingri\u0026rsquo; in India, stands out for its nutritional and medicinal benefits. It is rich in vitamin C, B-complex vitamins, and protein (1.6\u0026ndash;2.5%), contains essential minerals, and is cholesterol-free, making it easily digestible (Chauhan et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Its high potassium-to-sodium ratio supports cardiovascular health, while its folic acid content helps combat anemia (Ren et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Oyster mushrooms also exhibit antitumor, antioxidant, and antihyperglycemic properties, contributing to their global cultivation. Their adaptability to diverse agro-climatic conditions and ability to grow on agricultural waste further enhance their sustainability (Dimopoulou et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2025\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eMushroom cultivation of \u003cem\u003ePleurotus\u003c/em\u003e species holds promise for improving nutrition, health, and sustainability. However, the inadequate optimization of growth substrates remains a major challenge. The composition and quality of cultivation substrates strongly influence mycelial development, productivity, nutrient content, and secondary metabolite synthesis, while unstandardized use of agricultural residues can introduce mycotoxin contamination risks. To address these challenges, this study investigated how different agro-waste substrates, corn cob, sawdust, and compost waste, affect the growth performance, nutritional composition, and bioactive potential of \u003cem\u003ePleurotus pulmonarius\u003c/em\u003e and \u003cem\u003ePleurotus florida\u003c/em\u003e. It further evaluated morphological traits, proximate composition, aflatoxin contamination, and the mushrooms' antioxidant and antimicrobial capacities to ensure both quality and safety. This study was designed to establish safe and sustainable cultivation practices for \u003cem\u003ePleurotus\u003c/em\u003e species using agro-wastes, enhancing nutritional quality while promoting agricultural waste recycling. Overall, this work provides insight into substrate-dependent optimization for enhanced yield and metabolite production, supporting the advancement of \u003cem\u003ePleurotus\u003c/em\u003e cultivation as a reliable source of functional foods and natural therapeutic agents.\u003c/p\u003e"},{"header":"2.0 Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1 Materials\u003c/h2\u003e\u003cp\u003e\u003cem\u003ePleurotus pulmonarius\u003c/em\u003e and \u003cem\u003ePleurotus florida\u003c/em\u003e were obtained from a commercial supplier in Lagos State, Nigeria. The corn cobs were sourced from Malete, while sawdust was collected from Sawmill Road in Ilorin, Kwara State, under sterile conditions. Compost waste was also obtained from Malete. Furthermore, all mushroom cultivation procedures were conducted in the microbiology laboratory at Kwara State University, Malete. The cultivation was performed under ambient laboratory conditions without controlled temperature, humidity, or ventilation.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2 Cultivation Procedure of \u003cem\u003ePleurotus\u003c/em\u003e Species\u003c/h2\u003e\u003cp\u003eThe cultivation of \u003cem\u003ePleurotus pulmonarius\u003c/em\u003e and \u003cem\u003ePleurotus florida\u003c/em\u003e was carried out using three different substrates: corn cob, sawdust, and compost waste. The corn cob and sawdust substrates were thoroughly washed to remove dirt and debris, then moistened with distilled water to attain optimal moisture content. Compost waste, however, was sterilized directly without prior washing. All substrates were sterilized at 121\u0026deg;C for 15 minutes to eliminate microbial contaminants and subsequently allowed to cool to room temperature. After sterilization and cooling, 1 kg of each substrate was weighed and packed into heat-resistant polypropylene bags. Equal quantities of \u003cem\u003ePleurotus pulmonarius\u003c/em\u003e and \u003cem\u003ePleurotus florida\u003c/em\u003e spawn were inoculated into each substrate layer by layer under aseptic conditions. The bag openings were sealed with sterile cotton wool and covered with paper to prevent contamination during incubation. The inoculated bags containing corn cob, sawdust, and compost waste were incubated in a dark room at 20\u0026ndash;25\u0026deg;C for one week to initiate mycelial growth. Relative humidity was maintained between 70% and 85% by regularly spraying water on gunny bags covering the incubation chamber. Once full mycelial colonization and pinhead (primordia) formation were observed, small perforations were made in the bags to allow aeration and facilitate fruiting. During the fruiting phase, the bags were watered twice daily (morning and evening) to maintain adequate moisture, and diffused light was provided to promote proper fruit body development. Contaminated or poorly colonized bags were immediately discarded to prevent the spread of infection. Mature mushrooms were harvested by gently twisting the fruiting bodies from the substrate after six weeks (42 days) for corn cob and sawdust substrates, while those grown on compost waste were harvested after seven weeks (49 days) due to slower mycelial colonization and fruiting development. After harvesting, the bags were returned to the growing chamber for subsequent flushes. This cultivation procedure was adapted from Chai et al. (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), Jasinska and Siwulski (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), and Mishra et al. (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3 Growth and Yield Data Collection of \u003cem\u003ePleurotus\u003c/em\u003e Species\u003c/h2\u003e\u003cp\u003eGrowth and yield performance of \u003cem\u003ePleurotus pulmonarius\u003c/em\u003e and \u003cem\u003ePleurotus florida\u003c/em\u003e cultivated on the three substrates were evaluated following the methods of Chukwu et al. (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) and Bandura \u003cem\u003eet al\u003c/em\u003e. (2024). Data collection focused on the number, weight, and morphological dimensions of the fruiting bodies, as well as the time required for developmental stages. The time required for primordial initiation was determined by monitoring randomly selected bags from each substrate daily after incubation. The number of days from the completion of mycelial colonization to the appearance of the first primordia was recorded. The time required for harvest was measured from the day of primordial initiation to the maturity of the fruiting bodies, and mean values were computed. The total number of primordia and effective mature fruiting bodies per bag was counted and averaged across replicates. The fresh weight of individual fruiting bodies was measured using a digital weighing balance, and the average yield per bag was calculated. Morphological parameters such as height, stipe length, and cap diameter were measured in centimeters using a transparent ruler, and average values for each parameter were recorded for comparison among substrates.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4 Proximate Composition of Cultivated Mushroom\u003c/h2\u003e\u003cp\u003eThe proximate composition of the cultivated \u003cem\u003ePleurotus pulmonarius\u003c/em\u003e and \u003cem\u003ePleurotus florida\u003c/em\u003e samples grown on three different substrates, corn cob, sawdust, and compost waste, was analyzed to evaluate the nutritional composition of each species across the substrates. The analysis was conducted according to the standard AOAC procedures described by AOAC (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), determining key nutritional parameters including moisture content, crude protein, total ash, crude fibre, crude fat, and total carbohydrate.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.5 Determination of Aflatoxin Compounds in \u003cem\u003ePleurotus pulmonarius\u003c/em\u003e and \u003cem\u003ePleurotus florida\u003c/em\u003e using HPLC-FLD Analysis\u003c/h2\u003e\u003cdiv id=\"Sec8\" class=\"Section3\"\u003e\u003ch2\u003e2.5.1 Sample Preparation\u003c/h2\u003e\u003cp\u003eFresh fruiting bodies of \u003cem\u003ePleurotus pulmonarius\u003c/em\u003e and \u003cem\u003ePleurotus florida\u003c/em\u003e were harvested, cleaned to remove debris, and air-dried at 50\u0026deg;C until a constant weight was reached. The dried mushrooms were pulverized into fine powder using a laboratory mill and stored in amber glass bottles at 4\u0026deg;C until analysis.\u003c/p\u003e\u003cp\u003eFor extraction, 10 g of powdered sample was weighed into a 250 mL Erlenmeyer flask, and 50 mL of methanol\u0026ndash;acetonitrile\u0026ndash;water (60:30:10, v/v/v) was added. The mixture was vortexed for 5 min and mechanically shaken for 20 min at room temperature, then centrifuged at 5000 rpm for 10 min. The supernatant was collected for cleanup (Ahmed \u003cem\u003eet al\u003c/em\u003e., 2023).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section3\"\u003e\u003ch2\u003e2.5.2 Immunoaffinity Column Clean-Up\u003c/h2\u003e\u003cp\u003eTo purify the extract and remove matrix interferences, 20 mL of the clarified extract was diluted with 30 mL of phosphate-buffered saline (PBS, 0.1 M, pH 7.4) and filtered through glass microfiber paper. A 25 mL portion of the filtrate (equivalent to 1 g of sample) was passed through an AflaTest\u0026reg; immunoaffinity column (VICAM, USA) at a steady flow rate (~\u0026thinsp;1 mL/min) under gravity.\u003c/p\u003e\u003cp\u003eAfter sample loading, the column was washed with 5 mL PBS and 5 mL distilled water, then gently air-dried. The aflatoxins were eluted using 2 mL HPLC-grade methanol into amber vials. The eluate was filtered through a 0.45 \u0026micro;m PTFE syringe filter into HPLC vials without derivatization (Ahmed \u003cem\u003eet al\u003c/em\u003e., 2023).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section3\"\u003e\u003ch2\u003e2.5.3 HPLC-FLD Analysis\u003c/h2\u003e\u003cp\u003eQuantitative determination of aflatoxins was carried out using a Shimadzu LC-20A HPLC system equipped with a fluorescence detector (FLD). Separation was achieved on a reversed-phase C18 analytical column (150 mm \u0026times; 4.6 mm, 5 \u0026micro;m) maintained at 30\u0026deg;C.\u003c/p\u003e\u003cp\u003eThe mobile phase consisted of water\u0026ndash;methanol\u0026ndash;acetonitrile (60:20:20, v/v/v) under isocratic elution at a flow rate of 1.0 mL/min, and the injection volume was 20 \u0026micro;L. The detector was set at an excitation wavelength of 365 nm and an emission wavelength of 455 nm.\u003c/p\u003e\u003cp\u003eDistinct chromatographic peaks corresponding to aflatoxins B₂, B₁, and G₁ were observed at retention times of approximately 1.3, 1.9, and 3.1 minutes, respectively (Ahmed \u003cem\u003eet al\u003c/em\u003e., 2023).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section3\"\u003e\u003ch2\u003e2.5.4 Calibration and Quantification\u003c/h2\u003e\u003cp\u003eStandard stock solutions of aflatoxins B₁, B₂, G₁, and G₂ (Sigma-Aldrich, USA) were prepared in acetonitrile and diluted to working concentrations (0.2\u0026ndash;10 ng/mL). Calibration curves were plotted as peak area versus concentration for each aflatoxin, showing linearity with correlation coefficients (r\u0026sup2; \u0026gt;0.999).\u003c/p\u003e\u003cp\u003eSample concentrations were calculated from the standard curves and expressed as \u0026micro;g/kg (ppb) of dry mushroom weight. Peaks were identified by comparing retention times of samples with those of aflatoxin standards analyzed under identical conditions (Ahmed \u003cem\u003eet al\u003c/em\u003e., 2023).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section3\"\u003e\u003ch2\u003e2.5.5 Method Validation\u003c/h2\u003e\u003cp\u003eThe analytical method was validated for selectivity, linearity, precision, and recovery. Blank mushroom powder confirmed to be aflatoxin-free was spiked with standards at 2, 5, and 10 \u0026micro;g/kg levels and analyzed in triplicate. Mean recoveries ranged from 82\u0026ndash;108%, with relative standard deviations (RSD)\u0026thinsp;\u0026lt;\u0026thinsp;10%. Limits of detection (LOD) and quantification (LOQ) corresponded to signal-to-noise ratios of 3:1 and 10:1, respectively.\u003c/p\u003e\u003cp\u003eChromatograms were processed using Shimadzu LabSolutions software, and all results were expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation of triplicate determinations (Ahmed \u003cem\u003eet al\u003c/em\u003e., 2023).\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003e2.6. Qualitative Phytochemical Screening\u003c/h2\u003e\u003cp\u003eThe qualitative phytochemical screening of \u003cem\u003ePleurotus pulmonarius\u003c/em\u003e and \u003cem\u003ePleurotus florida\u003c/em\u003e samples for alkaloids, flavonoids, terpenoids, saponins, and tannin was carried out using standard methods described by Herawati et al. (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e\u003cdiv id=\"Sec14\" class=\"Section3\"\u003e\u003ch2\u003e2.6.1 Alkaloid Test\u003c/h2\u003e\u003cp\u003eThe presence of alkaloids in the extracts of \u003cem\u003ePleurotus pulmonarius\u003c/em\u003e and \u003cem\u003ePleurotus florida\u003c/em\u003e was determined using Dragendorff\u0026rsquo;s reagent. The reagent was prepared by dissolving 0.5 g of bismuth (III) nitrate in 6 mL of glacial acetic acid and 24 mL of distilled water (Solution I), while Solution II consisted of 12 g of potassium iodide dissolved in 30 mL of distilled water. Equal volumes (1 mL each) of the two solutions were mixed, followed by the addition of 2 mL of acetic acid and 10 mL of distilled water to obtain the working Dragendorff solution. For the test, 5 mL of each mushroom extract was mixed with 2 mL of potassium chloride (KCl) solution and 1 mL of Dragendorff reagent. The formation of an orange or reddish coloration indicated the presence of alkaloids.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section3\"\u003e\u003ch2\u003e2.6.2 Flavonoid Test\u003c/h2\u003e\u003cp\u003eFlavonoids were qualitatively identified by adding a few drops of 1% sodium hydroxide (NaOH) solution to 1 mL of the mushroom extract. The development of a bright yellow coloration that disappeared upon the addition of dilute hydrochloric acid (1% HCl) confirmed the presence of flavonoids in the extracts of \u003cem\u003eP. pulmonarius\u003c/em\u003e and \u003cem\u003eP. florida\u003c/em\u003e.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section3\"\u003e\u003ch2\u003e2.6.3 Saponin Test\u003c/h2\u003e\u003cp\u003eFor the detection of saponins, 1 mL of each mushroom extract, dissolved in acetone, was transferred into a test tube, and 10 mL of hot distilled water was added. The solution was allowed to cool and then shaken vigorously for about 10 seconds. The formation of a stable foam layer (1\u0026ndash;10 cm in height) that persisted for approximately 10 seconds and remained unchanged after the addition of a drop of 2N hydrochloric acid indicated the presence of saponins.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section3\"\u003e\u003ch2\u003e2.6.4 Tannin Test\u003c/h2\u003e\u003cp\u003eThe test was carried out by putting 10 mL of the mushroom extract solution into a test tube and adding 1% Pb (CH\u003csub\u003e3\u003c/sub\u003eCOO)\u003csub\u003e2\u003c/sub\u003e solution. Tannins are present when a yellow precipitate is formed during the reaction.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section3\"\u003e\u003ch2\u003e2.6.5 Triterpenoids Test\u003c/h2\u003e\u003cp\u003eIn this method, 1 mL of each \u003cem\u003ePleurotus pulmonarius\u003c/em\u003e and \u003cem\u003ePleurotus florida\u003c/em\u003e extract dissolved in acetone was placed in a test tube, followed by the addition of 10 drops of anhydrous acetic acid and 2 drops of concentrated sulfuric acid. The mixture was gently shaken and allowed to stand for a few minutes to observe any color change. The development of a red or purple coloration indicated the presence of triterpenoids, confirming that terpenoid compounds were present in the mushroom extracts.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003e2.7. Quantitative Phytochemical Analysis\u003c/h2\u003e\u003cp\u003eThe quantitative determination of phenolic compounds, flavonoids, terpenoids, and essential oils in \u003cem\u003ePleurotus pulmonarius\u003c/em\u003e and \u003cem\u003ePleurotus florida\u003c/em\u003e was carried out using standard spectrophotometric and hydrodistillation methods as described by Ogidi et al. (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) and Salachna et al. (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e\u003cdiv id=\"Sec20\" class=\"Section3\"\u003e\u003ch2\u003e2.7.1 Determination of Total Phenolic Content\u003c/h2\u003e\u003cp\u003eThe total phenolic content of \u003cem\u003ePleurotus pulmonarius\u003c/em\u003e and \u003cem\u003ePleurotus florida\u003c/em\u003e extracts was determined using a modified Folin\u0026ndash;Ciocalteu method. A 200 \u0026micro;L portion of each mushroom extract was mixed with 800 \u0026micro;L of Folin\u0026ndash;Ciocalteu reagent and 2 mL of 7.5% sodium carbonate (Na₂CO₃) solution. The mixture was diluted to a final volume of 7 mL with distilled water and incubated in the dark for 2 hours at room temperature. Absorbance was measured at 765 nm using a spectrophotometer. Gallic acid was used as the standard, and total phenolic content was expressed as milligrams of gallic acid equivalents per milliliter of extract (mg GAE/mL).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec21\" class=\"Section3\"\u003e\u003ch2\u003e2.7.2 Determination of Total Flavonoid Content\u003c/h2\u003e\u003cp\u003eThe total flavonoid content of \u003cem\u003ePleurotus pulmonarius\u003c/em\u003e and \u003cem\u003ePleurotus florida\u003c/em\u003e extracts was determined using the aluminum chloride colorimetric method. The mushroom extract was mixed with distilled water and sodium nitrite (NaNO₂) solution. After 6 minutes, aluminum chloride (AlCl₃) solution was added and allowed to stand for another 6 minutes, followed by the addition of sodium hydroxide (NaOH) solution. Distilled water was then added to make up the final volume, and the mixture was thoroughly mixed and left to stand for 15 minutes. The absorbance was measured at 510 nm using a spectrophotometer. Rutin was used as the standard, and total flavonoid content was calculated from the standard calibration curve and expressed as milligrams of rutin equivalents per gram of extract (mg RE/g).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec22\" class=\"Section3\"\u003e\u003ch2\u003e2.7.3 Determination of Total Terpenoid Content\u003c/h2\u003e\u003cp\u003eOne gram of each \u003cem\u003ePleurotus pulmonarius\u003c/em\u003e and \u003cem\u003ePleurotus florida\u003c/em\u003e extract was macerated in 50 mL of ethanol and filtered. To 2.5 mL of the filtrate, 2.5 mL of 5% aqueous phosphomolybdic acid solution and 2.5 mL of concentrated sulfuric acid (H₂SO₄) were added sequentially and mixed thoroughly. The mixture was allowed to stand for 30 minutes, after which the volume was made up to 12.5 mL with ethanol. The absorbance was then measured at 700 nm, and the total terpenoid content was determined accordingly.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec23\" class=\"Section3\"\u003e\u003ch2\u003e2.7.4 Determination of Essential Oil Content\u003c/h2\u003e\u003cp\u003eTwenty-five grams of dried samples of \u003cem\u003ePleurotus pulmonarius\u003c/em\u003e and \u003cem\u003ePleurotus florida\u003c/em\u003e were each mixed with 400 mL of distilled water and subjected to hydrodistillation for 3 hours using a Clevenger-type apparatus. The resulting essential oil was separated from the aqueous phase, dried over anhydrous sodium sulfate, filtered, and weighed. The oils were then stored in dark, airtight vials at 4\u0026deg;C until further analysis by GC\u0026ndash;MS.\u003c/p\u003e\u003cp\u003eThe essential oil content was calculated on a dry weight basis and expressed as a percentage (% w/w).\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec24\" class=\"Section2\"\u003e\u003ch2\u003e2.8. Antioxidant Activities of Cultivated \u003cem\u003ePleurotus pulmonarius\u003c/em\u003e and \u003cem\u003ePleurotus florida\u003c/em\u003e\u003c/h2\u003e\u003cdiv id=\"Sec25\" class=\"Section3\"\u003e\u003ch2\u003e2.8.1. Nitric Scavenging Assay\u003c/h2\u003e\u003cp\u003eThe nitric oxide scavenging activity of \u003cem\u003ePleurotus pulmonarius\u003c/em\u003e and \u003cem\u003ePleurotus florida\u003c/em\u003e extracts was determined based on the Griess reaction. Briefly, a reaction mixture (3 mL) containing 10 mM sodium nitroprusside and 1 mL of mushroom extract at varying concentrations (50\u0026ndash;100 mg/mL) was prepared in phosphate buffer (pH 7.4) and incubated at 25\u0026deg;C for 2.5 hours. After incubation, 1 mL of sulfanilic acid reagent was added to 0.5 mL of the reaction mixture and allowed to stand for 5 minutes, followed by the addition of 1 mL of 0.1% naphthyl ethylenediamine dihydrochloride. The resulting solution was incubated at room temperature for 30 minutes, and absorbance was measured at 540 nm using a UV\u0026ndash;visible spectrophotometer (Bristy et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Ascorbic acid served as the standard, and nitric oxide scavenging activity was expressed as percentage inhibition using the formula:\u003c/p\u003e\u003cp\u003e%NO scavenging= \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\:\\frac{AC-AS}{AC}\\times\\:100\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\u003cp\u003eWhere: AC absorbance of the negative control and AS absorbance of the sample.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec26\" class=\"Section3\"\u003e\u003ch2\u003e2.8.2. Ferric Scavenging Assay\u003c/h2\u003e\u003cp\u003eThe reducing power of \u003cem\u003ePleurotus pulmonarius\u003c/em\u003e and \u003cem\u003ePleurotus florida\u003c/em\u003e extract was measured as given by Boonsong et al. (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2016\u003c/span\u003e); Yusuf-Salihu et al. (\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Briefly, 1 ml of the extract of varying concentration (50\u0026ndash;100 mg/ml) with ascorbic acid serving as the standard reference, 2.5 ml of phosphate buffer (pH 6.6), and 2.5 ml of potassium ferricyanide (30 mM) were added and incubated at 50\u0026deg;C for 20 min. Then, 2.5 ml of trichloroacetic acid (600 mM) was added to the reaction mixture and centrifuged for 10 min at 3000 rpm. The upper layer of the solution (2.5 ml) was mixed with 2.5 ml of distilled water and 0.5 ml of FeCl\u003csub\u003e3\u003c/sub\u003e (6 mM), and absorbance was measured at 700 nm. Ascorbic acid was used as a standard. The percentage inhibition of ferric reducing power was calculated using the formula:\u003c/p\u003e\u003cp\u003e% Reducing Power = \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\:\\frac{AS-AB}{AC-\\text{A}\\text{B}}\\times\\:100\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eWhere\u003c/b\u003e:\u003c/p\u003e\u003cp\u003eAS\u0026thinsp;=\u0026thinsp;Absorbance of the sample extract\u003c/p\u003e\u003cp\u003eAC\u0026thinsp;=\u0026thinsp;Absorbance of the standard\u003c/p\u003e\u003cp\u003eAB\u0026thinsp;=\u0026thinsp;Absorbance of reagent blank (without sample or control)\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec27\" class=\"Section3\"\u003e\u003ch2\u003e2.8.3. DPPH (2,2-Diphenyl-1-picrylhydrazyl) Radical Scavenging Assay\u003c/h2\u003e\u003cp\u003eThe antioxidant activity of \u003cem\u003ePleurotus pulmonarius\u003c/em\u003e and \u003cem\u003ePleurotus florida\u003c/em\u003e extracts was evaluated using the DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging assay following Boonsong et al. (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) and Yusuf-Salihu et al. (\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). A 1 mM DPPH solution (3 mL) was mixed with 1 mL of mushroom extract at varying concentrations (50\u0026ndash;100 mg/mL) and incubated in the dark for 30 minutes at 27\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C. Ascorbic acid and methanol served as the positive control and blank, respectively. Absorbance was measured at 517 nm using a BIOBASE BK-D590 spectrophotometer, and the percentage inhibition of DPPH radicals was calculated using the formula:\u003c/p\u003e\u003cp\u003e%DPPH scavenging = \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\:\\frac{AC-AS}{AC}\\times\\:100\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\u003cp\u003eWhere: AC absorbance of the negative control and AS absorbance of the sample.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec28\" class=\"Section3\"\u003e\u003ch2\u003e2.8.4. Hydrogen Peroxide Scavenging Assay\u003c/h2\u003e\u003cp\u003eThe hydrogen peroxide scavenging assay was evaluated using the modified method of Bristy et al. (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). A solution of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e (40 mM) was prepared in a phosphate buffer of pH 7.4. The concentration of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e was determined by absorption using a spectrophotometer. 4 ml of different concentrations of 50,60,70,80,90, and 100 mg/ml of \u003cem\u003ePleurotus pulmonarius\u003c/em\u003e and \u003cem\u003ePleurotus florida\u003c/em\u003e, and distilled water was added to 0.60 ml, 40 mM of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e solution. The absorbance of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e at 230 nm was determined after 20 min against a blank solution (phosphate buffer with H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e), and ascorbic acid was used as the standard. The percentage of hydrogen peroxide scavenging by the \u003cem\u003ePleurotus pulmonarius\u003c/em\u003e and \u003cem\u003ePleurotus florida\u003c/em\u003e and standard compounds was calculated as follows:\u003c/p\u003e\u003cp\u003e% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e Scavenging = \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\frac{AC-AS}{AC}\\times\\:100\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\u003cp\u003eWhere: AC absorbance of the negative control and AS absorbance of the sample.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec29\" class=\"Section2\"\u003e\u003ch2\u003e2.9 Antimicrobial Activities of Cultivated \u003cem\u003ePleurotus pulmonarius\u003c/em\u003e and \u003cem\u003ePleurotus florida\u003c/em\u003e\u003c/h2\u003e\u003cdiv id=\"Sec30\" class=\"Section3\"\u003e\u003ch2\u003e2.9.1 Antibacterial Activity\u003c/h2\u003e\u003cp\u003eThe antibacterial activity of each mushroom aqueous extract was assessed using the broth culture method (Yusuf-Salihu et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Each bacterial strain was cultured in Nutrient Broth (NB) at 37\u0026deg;C for 24 hours, and the bacterial suspensions were adjusted to a concentration of 10\u003csup\u003e6\u003c/sup\u003e colony-forming units (cfu) per milliliter, equivalent to a 0.5 McFarland standard. \u003cem\u003eEscherichia coli, Klebsiella pneumoniae, Staphylococcus aureus\u003c/em\u003e, and \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e were used to inoculate 9 mL of peptone broth supplemented with 1 mL of \u003cem\u003ePleurotus pulmonarius\u003c/em\u003e and \u003cem\u003ePleurotus florida\u003c/em\u003e crude extracts at gradient concentrations of 50 mg/ml, 60 mg/ml, 70 mg/ml, 80 mg/ml, 90 mg/ml, and 100 mg/ml. The control group received no crude extract, while the positive control received 1 mL of chloramphenicol (30 \u0026micro;g/mL). The cultures were then incubated at 37\u0026deg;C for 24 hours. Subsequently, their optical density readings were measured at 600 nm, and the percentage growth inhibition was determined as follows:\u003c/p\u003e\u003cp\u003e% Growth inhibition = \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\frac{\\text{O}\\text{D}\\:\\text{c}\\text{o}\\text{n}\\text{t}\\text{r}\\text{o}\\text{l}-\\text{O}\\text{D}\\:\\text{t}\\text{e}\\text{s}\\text{t}}{\\text{O}\\text{D}\\:\\text{c}\\text{o}\\text{n}\\text{t}\\text{r}\\text{o}\\text{l}}\\times\\:100\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec31\" class=\"Section3\"\u003e\u003ch2\u003e2.9.2 Assay for Antifungal Activity\u003c/h2\u003e\u003cp\u003eThe antifungal activity of \u003cem\u003ePleurotus pulmonarius\u003c/em\u003e and \u003cem\u003ePleurotus florida\u003c/em\u003e extract was evaluated using the agar well diffusion method, with results expressed as zones of inhibition. Agar plates were inoculated with fungal strains \u003cem\u003eAspergillus fumigatus\u003c/em\u003e, \u003cem\u003eA. flavus\u003c/em\u003e, and \u003cem\u003eA. niger\u003c/em\u003e, and wells of 6 mm diameter were bored using a sterile cork borer. Each well was filled with 100 \u0026micro;L of the mushroom extract at concentrations of 50, 60, 70, 80, and 90 mg/mL, while sterilized distilled water served as the control. After incubation, the antifungal efficacy was determined by measuring the diameter of the inhibition zones around each well (Yusuf-Salihu et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2025\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec32\" class=\"Section2\"\u003e\u003ch2\u003e2.10. Statistical Analysis\u003c/h2\u003e\u003cp\u003eAll data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (n\u0026thinsp;=\u0026thinsp;3). A two-way ANOVA was used to assess the effects of mushroom species and substrate type on growth, yield, nutritional, and phytochemical parameters. A one-way ANOVA was applied to analyze concentration-dependent effects in antimicrobial and antioxidant assays. Tukey's HSD post-hoc test was used for multiple comparisons, with significance defined at p\u0026thinsp;\u0026lt;\u0026thinsp;0.05. Analyses were performed using IBM SPSS Statistics.\u003c/p\u003e\u003c/div\u003e"},{"header":"3.0 Results","content":"\u003cdiv id=\"Sec34\" class=\"Section2\"\u003e\u003ch2\u003e3.1. Morphological Growth of \u003cem\u003ePleurotus pulmonarius\u003c/em\u003e and \u003cem\u003ePleurotus florida\u003c/em\u003e on Different Substrates\u003c/h2\u003e\u003cp\u003eThe morphological growth characteristics of \u003cem\u003ePleurotus pulmonarius\u003c/em\u003e and \u003cem\u003ePleurotus florida\u003c/em\u003e cultivated on three different substrates sawdust, corn cob, and compost waste. Both species exhibited optimal growth on sawdust and corn cob substrates, which produced the highest total yield and well-developed morphological structures. On sawdust, \u003cem\u003eP. pulmonarius\u003c/em\u003e and \u003cem\u003eP. florida\u003c/em\u003e recorded mean yields of 181.07\u0026thinsp;\u0026plusmn;\u0026thinsp;0.82 g and 159.05\u0026thinsp;\u0026plusmn;\u0026thinsp;1.09 g, respectively, with large pileus diameters, longer stipes, and greater total height. Corn cob also supported vigorous growth, with \u003cem\u003eP. florida\u003c/em\u003e attaining a slightly higher total height (9.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.84 cm) than \u003cem\u003eP. pulmonarius\u003c/em\u003e (8.4\u0026thinsp;\u0026plusmn;\u0026thinsp;1.10 cm), indicating favorable substrate conditions. In contrast, compost waste resulted in the poorest growth performance for both species, with markedly smaller pilei, shorter stipes, and lower overall height, reflecting its limited nutrient availability and less supportive structure (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eMorphological growth of \u003cem\u003ePleurotus pulmonarius\u003c/em\u003e and \u003cem\u003ePleurotus florida\u003c/em\u003e on different substrates\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"9\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSubstrate\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e\u003cp\u003eTotal Yield per Bag (g)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e\u003cp\u003ePileus (Cap) Diameter (cm)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e\u003cp\u003eStipe Length (cm)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e\u003cp\u003eTotal Height (cm)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eP. pulmonarius\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003eP. florida\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003eP. pulmonarius\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cem\u003eP. florida\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u003cem\u003eP. pulmonarius\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u003cem\u003eP. florida\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u003cem\u003eP. pulmonarius\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e\u003cem\u003eP. florida\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eSawdust\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e181.07\u0026thinsp;\u0026plusmn;\u0026thinsp;0.82\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e159.05\u0026thinsp;\u0026plusmn;\u0026thinsp;1.09\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e5.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.85\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e6.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.78\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e4.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.65\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e4.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.73\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e7.3\u0026thinsp;\u0026plusmn;\u0026thinsp;1.02\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e8.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.95\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eCorn cob\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e158.12\u0026thinsp;\u0026plusmn;\u0026thinsp;1.47\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e170.40\u0026thinsp;\u0026plusmn;\u0026thinsp;1.38\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e6.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.85\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e6.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.92\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e5.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.80\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e5.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.68\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e8.4\u0026thinsp;\u0026plusmn;\u0026thinsp;1.10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e9.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.84\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eCompost waste\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e37.57\u0026thinsp;\u0026plusmn;\u0026thinsp;1.42\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e39.43\u0026thinsp;\u0026plusmn;\u0026thinsp;0.85\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e2.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.54\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e2.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.49\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e1.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.45\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e1.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.51\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e2.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.66\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e2.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.61\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec35\" class=\"Section2\"\u003e\u003ch2\u003e3.2. Yield Performance of \u003cem\u003ePleurotus pulmonarius\u003c/em\u003e and \u003cem\u003ePleurotus florida\u003c/em\u003e on Different Substrates\u003c/h2\u003e\u003cp\u003eThe yield performance of \u003cem\u003ePleurotus pulmonarius\u003c/em\u003e and \u003cem\u003ePleurotus florida\u003c/em\u003e cultivated on different substrates revealed notable variations in growth and fruiting behavior (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The shortest time required for primordial initiation was recorded on sawdust (19\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00 days for \u003cem\u003eP. pulmonarius\u003c/em\u003e and 20\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00 days for \u003cem\u003eP. florida\u003c/em\u003e), followed closely by corn cob (20\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00 and 18\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00 days, respectively). In contrast, mushrooms grown on compost waste exhibited the longest initiation period of 30\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00 days for both species. Similarly, the time to harvest was consistent across sawdust and corn cob substrates (42\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00 days) but extended to 49\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00 days on compost waste, reflecting slower mycelial colonization and fruiting. The number of total primordia and effective fruiting bodies followed a similar trend, with sawdust supporting the highest formation (24\u0026thinsp;\u0026plusmn;\u0026thinsp;1.00 and 21.67\u0026thinsp;\u0026plusmn;\u0026thinsp;1.53 primordia; 22\u0026thinsp;\u0026plusmn;\u0026thinsp;2.00 and 19.67\u0026thinsp;\u0026plusmn;\u0026thinsp;1.53 fruiting bodies for \u003cem\u003eP. pulmonarius\u003c/em\u003e and \u003cem\u003eP. florida\u003c/em\u003e, respectively). Corn cob showed moderate productivity, while compost waste produced the lowest number of primordia (10.50\u0026thinsp;\u0026plusmn;\u0026thinsp;0.50 and 6.67\u0026thinsp;\u0026plusmn;\u0026thinsp;1.15) and effective fruiting bodies (6.33\u0026thinsp;\u0026plusmn;\u0026thinsp;1.73 and 6.67\u0026thinsp;\u0026plusmn;\u0026thinsp;1.15) for the two species (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eYield Result of Fruiting Bodies of \u003cem\u003ePleurotus pulmonarius\u003c/em\u003e and \u003cem\u003ePleurotus florida\u003c/em\u003e on Different Substrates\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"9\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSubstrate\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e\u003cp\u003eTime required for primordial initiation (day)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e\u003cp\u003eTime required for harvest (days)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e\u003cp\u003eNumber of total primordial\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e\u003cp\u003eNumber of total effective fruiting bodies\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003ePleurotus pulmonarius\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003ePleurotus florida\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003ePleurotus pulmonarius\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cem\u003ePleurotus florida\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u003cem\u003ePleurotus pulmonarius\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u003cem\u003ePleurotus florida\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u003cem\u003ePleurotus pulmonarius\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e\u003cem\u003ePleurotus florida\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCorn cob\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e20\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e18.\u0026plusmn;0.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e42\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e42\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e20\u0026thinsp;\u0026plusmn;\u0026thinsp;0.816\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e13.83\u0026thinsp;\u0026plusmn;\u0026thinsp;1.52\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e13\u0026thinsp;\u0026plusmn;\u0026thinsp;2.65\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e7.33\u0026thinsp;\u0026plusmn;\u0026thinsp;1.09\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSawdust\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e19\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e20\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e42\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e42\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e24\u0026thinsp;\u0026plusmn;\u0026thinsp;1.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e21.67\u0026thinsp;\u0026plusmn;\u0026thinsp;1.53\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e22\u0026thinsp;\u0026plusmn;\u0026thinsp;2.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e19.67\u0026thinsp;\u0026plusmn;\u0026thinsp;1.53\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCompost\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e30\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e30\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e49\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e49\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e10.50\u0026thinsp;\u0026plusmn;\u0026thinsp;0.50\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e6.67\u0026thinsp;\u0026plusmn;\u0026thinsp;1.15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e6.33\u0026thinsp;\u0026plusmn;\u0026thinsp;1.73\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e6.67\u0026thinsp;\u0026plusmn;\u0026thinsp;1.15\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec36\" class=\"Section2\"\u003e\u003ch2\u003e3.3. Proximate Composition of \u003cem\u003ePleurotus pulmonarius\u003c/em\u003e and \u003cem\u003ePleurotus florida\u003c/em\u003e\u003c/h2\u003e\u003cp\u003eThe proximate composition of \u003cem\u003ePleurotus pulmonarius\u003c/em\u003e and \u003cem\u003ePleurotus florida\u003c/em\u003e was significantly affected by both substrate type and species, with a notable interaction between these factors (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Sawdust proved to be the most effective substrate for nutrient accumulation, supporting the highest levels of crude protein and ash. \u003cem\u003ePleurotus pulmonarius\u003c/em\u003e generally demonstrated a greater propensity for accumulating protein and ash than \u003cem\u003eP. florida\u003c/em\u003e when grown on sawdust and corn cob. Conversely, cultivation on compost waste resulted in a significantly diminished nutritional profile for both species, highlighting its inadequacy as a sole substrate. Furthermore, a strong species effect was evident in carbohydrate content, with \u003cem\u003eP. florida\u003c/em\u003e exhibiting a consistently and significantly higher concentration than \u003cem\u003eP. pulmonarius\u003c/em\u003e across all substrates\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eProximate Composition of \u003cem\u003ePleurotus pulmonarius\u003c/em\u003e and \u003cem\u003ePleurotus florida\u003c/em\u003e on Corn Cob, Sawdust, and Compost Waste\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"8\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSubstrate\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMushroom Species\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eMoisture Content (%)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eCrude Protein (%)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eTotal Ash (%)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eCrude Fibre (%)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003eCrude Fat (%)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c8\"\u003e\u003cp\u003eCarbohydrate (%)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eCorn cob\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eP. florida\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e85.56\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e3.06\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e0.98\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e\u003cp\u003e1.51\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e\u003cp\u003e2.53\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c8\"\u003e\u003cp\u003e6.35\u0026thinsp;\u0026plusmn;\u0026thinsp;0.14\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eP. pulmonarius\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e88.66\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e3.14\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e1.50\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e\u003cp\u003e1.72\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e\u003cp\u003e2.55\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c8\"\u003e\u003cp\u003e2.45\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eSawdust\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eP. florida\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e84.42\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e3.22\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e1.12\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e\u003cp\u003e1.63\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e\u003cp\u003e2.61\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c8\"\u003e\u003cp\u003e6.08\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eP. pulmonarius\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e87.12\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e3.31\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e1.61\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e\u003cp\u003e1.84\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e\u003cp\u003e2.62\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c8\"\u003e\u003cp\u003e2.23\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eCompost waste\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eP. florida\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e82.85\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e2.48\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e0.83\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e\u003cp\u003e1.24\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e\u003cp\u003e2.02\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c8\"\u003e\u003cp\u003e4.62\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eP. pulmonarius\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e85.90\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e2.68\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e1.18\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e\u003cp\u003e1.36\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e\u003cp\u003e2.12\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c8\"\u003e\u003cp\u003e1.54\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec37\" class=\"Section2\"\u003e\u003ch2\u003e3.4 Mycotoxin Analysis\u003c/h2\u003e\u003cdiv id=\"Sec38\" class=\"Section3\"\u003e\u003ch2\u003e3.4.1 Quantitative Analysis of Aflatoxin Compounds in \u003cem\u003ePleurotus pulmonarius\u003c/em\u003e Sample\u003c/h2\u003e\u003cp\u003eThe analysis of the \u003cem\u003ePleurotus pulmonarius\u003c/em\u003e sample revealed the presence of several aflatoxin compounds, as shown in the chromatogram (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) and summarized in Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. Aflatoxin B₁ was detected at the highest concentration of 6.7152 ppb, followed by Aflatoxin B₂ (1.2025 ppb) and Aflatoxin G₁ (0.9588 ppb). Additional minor peaks were detected, likely representing residual matrix components or degradation products. The concentrations of all detected aflatoxins were below the maximum regulatory limits for food safety.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eQuantitative Analysis of Aflatoxin Compounds in \u003cem\u003ePleurotus pulmonarius\u003c/em\u003e Sample\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"6\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePeak No.\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePeak ID\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eRet Time (min)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eHeight\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eArea\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eConcentration (ppb)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eSolvent front\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.907\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e78.886\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e1084.386\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eAflatoxin B2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e1.332\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e1036.629\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e7272.236\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e1.2025\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eUnidentified\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e1.640\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e56.971\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e203.329\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.0255\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eAflatoxin B1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e1.948\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e74.679\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e532.400\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e6.7152\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eUnidentified\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e2.148\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e97.045\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e961.400\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.5182\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eAflatoxin G1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e3.157\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e133.190\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e1504.000\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.9588\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eUnidentified\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e4.823\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e44.421\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e225.800\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.1162\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eNote: ppb\u0026thinsp;=\u0026thinsp;\u0026micro;g/kg dry weight. The solvent front is not a compound and is not quantified.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec39\" class=\"Section3\"\u003e\u003ch2\u003e3.4.2 Quantitative Analysis of Aflatoxin Compounds in \u003cem\u003ePleurotus florida\u003c/em\u003e Sample\u003c/h2\u003e\u003cp\u003eThe HPLC analysis of the \u003cem\u003ePleurotus florida\u003c/em\u003e sample confirmed the presence of aflatoxins, as detailed in Table\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e and shown in the chromatogram (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The sample contained Aflatoxin B₁ at 4.1774 ppb, Aflatoxin G₁ at 2.7558 ppb, and Aflatoxin B₂ at 0.4163 ppb. Similar to the \u003cem\u003eP. pulmonarius\u003c/em\u003e sample, multiple unidentified peaks were present. The total aflatoxin load was lower in \u003cem\u003eP. florida\u003c/em\u003e compared to \u003cem\u003eP. pulmonarius\u003c/em\u003e..\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab5\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eQuantitative Analysis of Aflatoxin Compounds in \u003cem\u003ePleurotus florid\u003c/em\u003e Sample\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"6\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePeak No.\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePeak ID\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eRet Time (min)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eHeight\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eArea\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eConcentration (ppb)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eSolvent front\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.132\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e35.884\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e392.150\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eAflatoxin B2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e1.115\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e55.286\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e293.400\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.4163\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eAflatoxin B1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e1.365\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e450.000\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e3545.800\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e4.1774\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eUnidentified\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e1.498\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e460.000\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e2101.300\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e1.6984\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eAflatoxin G1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e2.107\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e118.059\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e2989.400\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e2.7558\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eUnidentified\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e2.448\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e112.342\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e1234.000\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.8062\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eUnidentified\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e3.390\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e103.882\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e485.450\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.4577\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eUnidentified\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e3.573\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e59.240\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e791.650\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.5910\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eUnidentified\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e4.865\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e73.375\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e750.700\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.4956\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec40\" class=\"Section2\"\u003e\u003ch2\u003e3.5 Qualitative Screening of Phytochemical Compounds in \u003cem\u003ePleurotus pulmonarius\u003c/em\u003e and \u003cem\u003ePleurotus florida\u003c/em\u003e Cultivated on Different Substrates\u003c/h2\u003e\u003cp\u003eThe qualitative screening of anti-nutrients in \u003cem\u003ePleurotus pulmonarius\u003c/em\u003e and \u003cem\u003ePleurotus florida\u003c/em\u003e cultivated on sawdust, corn cob, and compost substrates revealed distinct variations in the presence of alkaloids, flavonoids, terpenoids, saponins, and tannin. Overall, both mushroom species exhibited moderate to high levels of alkaloids and flavonoids, with \u003cem\u003eP. florida\u003c/em\u003e grown on sawdust showing the highest flavonoid (+++) and tannin (+++) contents, while \u003cem\u003eP. Pulmonarius\u003c/em\u003e on sawdust recorded the highest alkaloid (+++) and saponin (++) concentrations. Samples grown on corn cob and compost substrates displayed relatively lower but consistent levels of these bioactive compounds, suggesting that the substrate type significantly influences the qualitative distribution of anti-nutritional components in both mushroom species.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab6\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 6\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eQualitative Phytochemical Screening of \u003cem\u003ePleurotus pulmonarius\u003c/em\u003e and \u003cem\u003ePleurotus florida\u003c/em\u003e cultivated on different substrates\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"7\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSubstrate\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMushroom species\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eAlkaloids\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eFlavonoids\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eTerpenoids\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eSaponins\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003eTannin\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eSawdust\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eP. florida\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e++\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003e+++\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003e+\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u003cb\u003e+\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u003cb\u003e+++\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eP. pulmonarius\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e+++\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003e++\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003e+\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u003cb\u003e++\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u003cb\u003e+\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eCorn cob\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eP. florida\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e++\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003e+++\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003e++\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u003cb\u003e+\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u003cb\u003e++\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eP. pulmonarius\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e++\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003e++\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003e+\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u003cb\u003e++\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u003cb\u003e+++\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eCompost\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eP. florida\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e+\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003e++\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003e+\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u003cb\u003e+\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u003cb\u003e++\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eP. pulmonarius\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e+\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003e+\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003e+\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u003cb\u003e++\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u003cb\u003e+\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eWhere (+)\u0026thinsp;=\u0026thinsp;low presence, (++)\u0026thinsp;=\u0026thinsp;moderate presence, (+++)\u0026thinsp;=\u0026thinsp;high presence of respective phytochemicals.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec41\" class=\"Section2\"\u003e\u003ch2\u003e3.6 Quantitative Composition of \u003cem\u003ePleurotus pulmonarius\u003c/em\u003e and \u003cem\u003ePleurotus florida\u003c/em\u003e Cultivated on Different Substrates\u003c/h2\u003e\u003cp\u003eThe quantitative analysis of \u003cem\u003ePleurotus pulmonarius\u003c/em\u003e and \u003cem\u003ePleurotus florida\u003c/em\u003e cultivated on sawdust, corn cob, and compost substrates revealed notable variations in the concentrations of phenolic compounds, flavonoids, terpenoids, and essential oils. \u003cem\u003eP. florida\u003c/em\u003e cultivated on sawdust recorded the highest phenolic (622.94 mg/kg) and flavonoid (27.01 mg/kg) contents, while \u003cem\u003eP. pulmonarius\u003c/em\u003e on the same substrate showed slightly lower values. Samples grown on corn cob exhibited relatively high concentrations of bioactive compounds, with \u003cem\u003eP. florida\u003c/em\u003e showing the highest flavonoid (30.00 mg/kg) and essential oil (16.50 mg/100 g) levels among all. Conversely, mushrooms grown on compost recorded the lowest phenolic and flavonoid contents, indicating that the type of substrate significantly affects the accumulation of secondary metabolites and essential oils in both \u003cem\u003ePleurotus\u003c/em\u003e species.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab7\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 7\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eQuantitative phytochemical composition of \u003cem\u003ePleurotus pulmonarius\u003c/em\u003e and \u003cem\u003ePleurotus florida\u003c/em\u003e cultivated on different substrates\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"6\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSubstrate\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMushroom species\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003ePhenolic compounds (mg/kg)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eFlavonoids (mg/kg)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eTerpenoids (mg/kg)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eEssential oils (mg/100 g)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eSawdust\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eP. florida\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e622.94\u0026thinsp;\u0026plusmn;\u0026thinsp;0.26\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e27.01\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e1.66\u0026thinsp;\u0026plusmn;\u0026thinsp;0.40\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e\u003cp\u003e17.02\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eP. pulmonarius\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e601.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.55\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e25.01\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e1.84\u0026thinsp;\u0026plusmn;\u0026thinsp;0.60\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e\u003cp\u003e15.02\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eCorn cob\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eP. florida\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e600.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.20\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e30.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e2.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.35\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e\u003cp\u003e16.50\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eP. pulmonarius\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e510.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e25.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e1.80\u0026thinsp;\u0026plusmn;\u0026thinsp;0.30\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e\u003cp\u003e17.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eCompost\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eP. florida\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e550.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.30\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e20.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e1.60\u0026thinsp;\u0026plusmn;\u0026thinsp;0.25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e\u003cp\u003e15.50\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eP. pulmonarius\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e539.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.35\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e18.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e1.54\u0026thinsp;\u0026plusmn;\u0026thinsp;0.28\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e\u003cp\u003e15.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.18\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec42\" class=\"Section2\"\u003e\u003ch2\u003e3.7 Antioxidant Activities\u003c/h2\u003e\u003cdiv id=\"Sec43\" class=\"Section3\"\u003e\u003ch2\u003e3.7.1 Ferric Reducing Activity of \u003cem\u003ePleurotus florida\u003c/em\u003e and \u003cem\u003ePleurotus pulmonarius\u003c/em\u003e\u003c/h2\u003e\u003cp\u003eThe ferric reducing antioxidant power (FRAP) of \u003cem\u003ePleurotus pulmonarius\u003c/em\u003e and \u003cem\u003ePleurotus florida\u003c/em\u003e increased progressively with concentration from 50 to 100 mg/mL, showing a dose-dependent response compared to the standard (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). At lower concentrations, both species exhibited moderate reducing ability, which intensified at higher concentrations. \u003cem\u003eP. florida\u003c/em\u003e consistently showed higher ferric reducing activity than \u003cem\u003eP. pulmonarius\u003c/em\u003e, with maximum values of 85% and 78% respectively at 90 mg/mL, while the standard reached 91%.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec44\" class=\"Section3\"\u003e\u003ch2\u003e3.7.2. Nitric Oxide Assay Pleurotus florida and \u003cem\u003ePleurotus pulmonarius\u003c/em\u003e\u003c/h2\u003e\u003cp\u003eThe nitric oxide (NO) scavenging activity of \u003cem\u003ePleurotus pulmonarius\u003c/em\u003e and \u003cem\u003ePleurotus florida\u003c/em\u003e increased progressively with concentration from 50 to 100 mg/mL, indicating a clear dose-dependent antioxidant response (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Both species demonstrated appreciable nitric oxide inhibition comparable to the standard, though \u003cem\u003eP. florida\u003c/em\u003e consistently exhibited slightly higher activity across all concentrations. At 100 mg/mL, \u003cem\u003eP. florida\u003c/em\u003e recorded 84% inhibition, while \u003cem\u003eP. pulmonarius\u003c/em\u003e achieved 81%, compared to 92% for the standard.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec45\" class=\"Section3\"\u003e\u003ch2\u003e3.7.3 DPPH Radical Scavenging Activity of \u003cem\u003ePleurotus pulmonarius\u003c/em\u003e and \u003cem\u003ePleurotus florida\u003c/em\u003e\u003c/h2\u003e\u003cp\u003eThe DPPH radical scavenging activity of \u003cem\u003ePleurotus pulmonarius\u003c/em\u003e and \u003cem\u003ePleurotus florida\u003c/em\u003e increased progressively with concentration from 50 to 100 mg/mL, indicating a clear dose-dependent antioxidant response (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Both species showed appreciable DPPH scavenging ability comparable to the standard, with \u003cem\u003eP. florida\u003c/em\u003e consistently exhibiting slightly higher activity at all concentrations. At 100 mg/mL, \u003cem\u003eP. florida\u003c/em\u003e recorded 89% inhibition, while \u003cem\u003eP. pulmonarius\u003c/em\u003e achieved 84%, compared to 95% for the standard.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec46\" class=\"Section3\"\u003e\u003ch2\u003e3.7.4 Hydrogen Peroxide Scavenging Activity of \u003cem\u003ePleurotus pulmonarius\u003c/em\u003e and \u003cem\u003ePleurotus florida\u003c/em\u003e\u003c/h2\u003e\u003cp\u003eThe hydrogen peroxide (H₂O₂) scavenging activity of \u003cem\u003ePleurotus pulmonarius\u003c/em\u003e and \u003cem\u003ePleurotus florida\u003c/em\u003e increased progressively with concentration from 50 to 100 mg/mL, showing a clear dose-dependent antioxidant response (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Both species exhibited considerable hydrogen peroxide scavenging ability comparable to the standard, with \u003cem\u003eP. florida\u003c/em\u003e consistently displaying slightly higher activity across all concentrations. At 100 mg/mL, \u003cem\u003eP. florida\u003c/em\u003e recorded 84% inhibition, while \u003cem\u003eP. pulmonarius\u003c/em\u003e achieved 80%, compared to 92% for the standard.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec47\" class=\"Section2\"\u003e\u003ch2\u003e3.8. Antimicrobial Activity of \u003cem\u003ePleurotus\u003c/em\u003e Extracts\u003c/h2\u003e\u003cdiv id=\"Sec48\" class=\"Section3\"\u003e\u003ch2\u003e3.8.1 Antibacterial Activity of \u003cem\u003ePleurotus\u003c/em\u003e Extracts\u003c/h2\u003e\u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e, the antibacterial activity of \u003cem\u003ePleurotus pulmonarius\u003c/em\u003e and \u003cem\u003ePleurotus florida\u003c/em\u003e extracts increased progressively with higher extract concentrations, demonstrating a dose-dependent inhibitory effect against all tested bacterial isolates. In \u003cem\u003eP. pulmonarius\u003c/em\u003e extract (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e), \u003cem\u003eStaphylococcus aureus\u003c/em\u003e exhibited the highest inhibition (78.69% at 100 mg/mL), followed by \u003cem\u003eEscherichia coli\u003c/em\u003e (70.32%), \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e (68.76%), and \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e (65.08%). Similarly, \u003cem\u003eP. florida\u003c/em\u003e extract (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e) showed maximum inhibition against \u003cem\u003eS. aureus\u003c/em\u003e (86.2%) and \u003cem\u003eE. coli\u003c/em\u003e (85.3%) at 100 mg/mL, while \u003cem\u003eK. pneumoniae\u003c/em\u003e and \u003cem\u003eP. aeruginosa\u003c/em\u003e displayed comparatively lower inhibition values. Both extracts exhibited strong antibacterial efficacy when compared with the positive control and no inhibition with the negative control.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec49\" class=\"Section3\"\u003e\u003ch2\u003e3.8.2 Antifungal Activity of \u003cem\u003ePleurotus florida\u003c/em\u003e Extract Against Fungal Strains\u003c/h2\u003e\u003cp\u003eThe aqueous extracts of both \u003cem\u003ePleurotus pulmonarius\u003c/em\u003e and \u003cem\u003ePleurotus florida\u003c/em\u003e exhibited notable, concentration-dependent antifungal activity against \u003cem\u003eAspergillus fumigatus\u003c/em\u003e, \u003cem\u003eA. flavus\u003c/em\u003e, and \u003cem\u003eA. niger\u003c/em\u003e, as shown in Tables\u0026nbsp;\u003cspan refid=\"Tab8\" class=\"InternalRef\"\u003e8\u003c/span\u003e and \u003cspan refid=\"Tab9\" class=\"InternalRef\"\u003e9\u003c/span\u003e. For both mushroom species, the zones of inhibition increased progressively with higher extract concentrations, confirming the presence of potent antifungal bioactive compounds. This inhibitory effect was visually evident in the plate assays, where distinct, clear zones surrounded the wells containing the extracts of \u003cem\u003eP. pulmonarius\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e) and \u003cem\u003eP. florida\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eAmong the tested fungi, \u003cem\u003eA. fumigatus\u003c/em\u003e showed the highest susceptibility to both extracts. The extract from \u003cem\u003eP. pulmonarius\u003c/em\u003e exhibited its strongest inhibition (33 mm) against \u003cem\u003eA. fumigatus\u003c/em\u003e at 90 mg/mL, followed by \u003cem\u003eA. niger\u003c/em\u003e (30 mm) and \u003cem\u003eA. flavus\u003c/em\u003e (20 mm), as presented in Table\u0026nbsp;\u003cspan refid=\"Tab8\" class=\"InternalRef\"\u003e8\u003c/span\u003e. Similarly, \u003cem\u003eP. florida\u003c/em\u003e extract demonstrated superior antifungal activity, maintaining the largest inhibition zone of 35 mm against \u003cem\u003eA. fumigatus\u003c/em\u003e across all concentrations tested, while \u003cem\u003eA. niger\u003c/em\u003e and \u003cem\u003eA. flavus\u003c/em\u003e showed inhibition zones of 31 mm and 18 mm, respectively, at 90 mg/mL (Table\u0026nbsp;\u003cspan refid=\"Tab9\" class=\"InternalRef\"\u003e9\u003c/span\u003e). Overall, \u003cem\u003eP. florida\u003c/em\u003e extract displayed a slightly higher antifungal potency than \u003cem\u003eP. pulmonarius\u003c/em\u003e, particularly against \u003cem\u003eA. fumigatus\u003c/em\u003e and \u003cem\u003eA. niger\u003c/em\u003e, indicating a stronger inhibitory potential.\u003c/p\u003e\u003cp\u003e.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab8\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 8\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eTable Showing Antifungal Activity on Fungal Strains using \u003cem\u003ePleurotus pulmonarius\u003c/em\u003e extract\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"6\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTest organism\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"5\" nameend=\"c6\" namest=\"c2\"\u003e\u003cp\u003eZone of Inhibition (mm)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003e50 mg/ml\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003e60 mg/ml\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003e70 mg/ml\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003e80 mg/ml\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003e90 mg/ml\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eA.fumigatus\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e20\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e21\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e31\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e33\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eA.flavus\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e17\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e17\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e21.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e20\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eA.niger\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e22\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e27\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e27\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e30\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab9\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 9\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eTable Showing Antifungal Activity on Fungal Strains Using \u003cem\u003ePleurotus florida\u003c/em\u003e extract\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"6\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTest organism\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"5\" nameend=\"c6\" namest=\"c2\"\u003e\u003cp\u003eZone of Inhibition (mm)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003e50 mg/ml\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003e60 mg/ml\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003e70 mg/ml\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003e80 mg/ml\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003e90 mg/ml\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eA.fumigatus\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e33\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e35\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e35\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e35\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e35\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eA.flavus\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e12\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e17\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e18\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eA.niger\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e10.50\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e24\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e29\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e31\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e"},{"header":"Discussions","content":"\u003cp\u003eThe superior morphological development of \u003cem\u003ePleurotus pulmonarius\u003c/em\u003e and \u003cem\u003eP. florida\u003c/em\u003e on sawdust and corn cob substrates reflects the influence of substrate composition on mushroom growth and yield. The high productivity and well-formed fruiting bodies obtained from these substrates can be attributed to their favorable physical and chemical properties particularly their porous structure, balanced carbon-to-nitrogen ratio, and high lignocellulosic content which enhance aeration, moisture retention, enzymatic activity, and mycelial colonization. The fibrous texture of sawdust and corn cob provides an ideal matrix for mycelial penetration and efficient degradation of organic material, supporting the formation of larger pilei, longer stipes, and taller fruiting bodies as observed in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. In contrast, compost waste exhibited poor performance, likely due to its compact structure, reduced aeration, and nutrient imbalance, which limited mycelial spread and morphological differentiation. These findings align with the observations of Raman et al. (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), who reported improved yield and fruit body morphology of \u003cem\u003ePleurotus\u003c/em\u003e species grown on corn- and wood-based lignocellulosic substrates. Similarly, Diamantopoulou et al. (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) and Han et al. (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) emphasized that the physicochemical composition of substrates especially the quality of lignocellulosic material and carbon-to-nitrogen balance plays a decisive role in determining the growth rate and morphological traits of cultivated mushrooms. Overall, the results of this study show the critical role of substrate selection in optimizing mushroom cultivation, with corn cob and sawdust identified as the most effective substrates for achieving superior morphological and yield characteristics in \u003cem\u003ePleurotus\u003c/em\u003e species.\u003c/p\u003e\u003cp\u003eThe observed variations in the yield performance of \u003cem\u003ePleurotus pulmonarius\u003c/em\u003e and \u003cem\u003eP. florida\u003c/em\u003e across the three substrates reflect the combined effects of nutrient composition, substrate texture, and aeration capacity on fungal colonization and fruiting efficiency. The superior yield obtained on sawdust and corn cob suggests that these substrates provided optimal physicochemical conditions for mycelial development and primordia formation. Sawdust, with its fine and fibrous texture, offers a stable and moisture-retentive environment that supports dense mycelial networks and abundant primordia, leading to higher numbers of effective fruiting bodies. Conversely, the porous structure and balanced carbon-to-nitrogen ratio of corn cob likely facilitated rapid nutrient diffusion and aeration, which favored faster primordial initiation and fruit body expansion despite slightly lower total yields than sawdust. The delayed initiation and reduced yield observed on compost waste can be attributed to its heterogeneous composition, poor structural integrity, and possible microbial competition, which collectively impede mycelial penetration and nutrient uptake. These findings align with the observations of Castorina et al. (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), who reported enhanced fruiting and shorter initiation times for \u003cem\u003ePleurotus\u003c/em\u003e species cultivated on maize cob due to its superior porosity and nutrient balance. Similarly, Argaw et al. (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) noted that sawdust substrates promote larger and thicker pilei across successive flushes, highlighting their structural stability and sustained nutrient availability, even when overall yield is moderately lower than on leaf-based substrates. The comparatively poor performance of compost waste contrasts with the results of Sultana et al. (\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), who demonstrated that yield improvements in compost-based systems depend on organic enrichment with additives such as poultry manure or oil cakes. Overall, the present findings emphasize the importance of substrate-specific optimization strategies in \u003cem\u003ePleurotus\u003c/em\u003e cultivation, confirming that sawdust and corn cob provide superior growth environments that enhance both yield and morphological quality, whereas compost waste remains the least effective substrate for productive mushroom cultivation.\u003c/p\u003e\u003cp\u003eThe detection of aflatoxin compounds in \u003cem\u003ePleurotus pulmonarius\u003c/em\u003e and \u003cem\u003ePleurotus florida\u003c/em\u003e indicates possible uptake or residual contamination from the substrates used during cultivation, as aflatoxins are secondary metabolites typically produced by \u003cem\u003eAspergillus\u003c/em\u003e species that may persist in agricultural residues even after sterilization. The differential aflatoxin profiles observed between the two species, with each accumulating specific aflatoxin analogs at varying levels, suggest species-specific variations in the uptake, bioaccumulation, or metabolic detoxification mechanisms. This finding is supported by Hainghumbi (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), who demonstrated that mycotoxin contamination in edible mushrooms was directly influenced by the natural substrates on which they grew. Importantly, the concentrations of all detected aflatoxins in both species remained below the maximum regulatory limits for food safety, indicating that the mushrooms are safe for consumption. Recent studies by Arimboor \u003cem\u003eet al.\u003c/em\u003e (2024) and Zapaśnik et al. (\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2025\u003c/span\u003e) also suggest that \u003cem\u003ePleurotus\u003c/em\u003e species possess intrinsic ligninolytic enzymes, which may enable the partial degradation of aflatoxins through oxidative and hydrolytic pathways, highlighting their potential for natural mycotoxin detoxification. Overall, the results underscore that while trace aflatoxin residues can occur in cultivated mushrooms, they are likely substrate-derived, and careful substrate selection and proper sterilization remain essential for ensuring food safety.\u003c/p\u003e\u003cp\u003eThe variations in the qualitative and quantitative composition of anti-nutritional and bioactive compounds in \u003cem\u003ePleurotus pulmonarius\u003c/em\u003e and \u003cem\u003ePleurotus florida\u003c/em\u003e cultivated on different substrates can be attributed to differences in substrate composition, nutrient availability, and metabolic adaptability of each mushroom species. Sawdust supported the highest accumulation of alkaloids, flavonoids, and phenolic compounds, likely due to its richness in lignin and cellulose, which induces oxidative enzyme activity and stimulates secondary metabolite synthesis. In contrast, corn cobs, with their balanced carbon-to-nitrogen ratio and moderate lignocellulosic content, promote relatively high levels of flavonoids and essential oils, reflecting their efficient nutrient exchange and aeration properties that favor the biosynthesis of antioxidant compounds. Compost waste, however, exhibited lower concentrations of these metabolites, possibly because of nutrient degradation and lower substrate stability after decomposition, which limits the metabolic pathways responsible for bioactive compound production. These observations align with the findings of Eze et al. (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), who reported that variations in substrate composition, such as maize kernel, dry banana leaf, and empty palm fruit bunch, significantly affect the yield, nutritional profile, and phytochemical accumulation in \u003cem\u003ePleurotus pulmonarius\u003c/em\u003e. Likewise, Baptista et al. (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) demonstrated that the biochemical composition of \u003cem\u003eLentinula edodes\u003c/em\u003e spent mushroom substrate (SMS) and the extraction conditions markedly influence the concentration of phenolic compounds, antioxidant capacity, and antibacterial activity. Similarly, Verduzco-Oliva and Gutierrez-Uribe \u003cem\u003eet al.\u003c/em\u003e (2020) noted who demonstrated that during solid-state fermentation of plant-based lignocellulosic residues, fungi secrete lignocellulolytic enzymes such as ligninases, cellulases, and hemicellulases that break down the fiber structure, thereby releasing phenolic compounds and stimulating the biosynthesis of additional bioactive metabolites. This indicates that lignocellulosic substrates like sawdust can enhance phenolic and flavonoid synthesis through their structural complexity and availability of lignin-derived precursors. Overall, this study reinforces that the type and quality of substrate not only determine mushroom yield but also significantly affect their phytochemical richness, with sawdust and corn cob emerging as superior substrates for producing nutritionally and medicinally valuable \u003cem\u003ePleurotus\u003c/em\u003e mushrooms.\u003c/p\u003e\u003cp\u003eThe antioxidant activities exhibited by \u003cem\u003ePleurotus florida\u003c/em\u003e and \u003cem\u003ePleurotus pulmonarius\u003c/em\u003e are attributed to their rich composition of phenolic compounds, flavonoids, and essential oils, which act as potent free radical scavengers and reducing agents. The consistently higher antioxidant performance of \u003cem\u003eP. florida\u003c/em\u003e across the ferric reducing, nitric oxide, DPPH, and hydrogen peroxide assays suggests that this species possesses a more efficient redox system, possibly due to higher concentrations of bioactive metabolites derived from substrate interactions. The use of sawdust and corn cob as substrates likely contributed to this enhanced antioxidant capacity, as both materials are rich in lignin and cellulose, which stimulate the production of secondary metabolites involved in oxidative defense. Compost waste, in contrast, may provide fewer biochemical precursors for phenolic synthesis, resulting in comparatively weaker antioxidant responses. Similarly, Diamantopoulou et al. (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) demonstrated that the antioxidant activity of \u003cem\u003ePleurotus\u003c/em\u003e mushrooms strongly correlates with substrate composition and phenolic content, while Rusu \u003cem\u003eet al\u003c/em\u003e. (2022\u003cb\u003e)\u003c/b\u003e found that supplementing cultivation substrates with brewery-spent grains significantly enhanced protein, flavonoid, and phenolic levels, leading to improved antioxidant activity. Similarly, Kumla et al. (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) reported that mushrooms cultivated on lignocellulosic agro-industrial wastes exhibit enhanced antioxidant activity due to increased production of lignocellulolytic enzymes that stimulate the synthesis of phenolic and flavonoid antioxidants. Overall, this study indicates that substrate type and bioactive compound composition significantly influence antioxidant potential, with \u003cem\u003eP. florida\u003c/em\u003e particularly when cultivated on nutrient-balanced substrates like corn cob and sawdust, showing greater capacity to neutralize free radicals and mitigate oxidative stress.\u003c/p\u003e\u003cp\u003eThe antimicrobial efficacy demonstrated by the \u003cem\u003ePleurotus\u003c/em\u003e extracts in this study shows the growing recognition of edible mushrooms as reservoirs of bioactive compounds with therapeutic potential. Rather than being an incidental property, this antimicrobial activity likely reflects a complex interplay between species-specific metabolic pathways and substrate-induced biochemical modulation. The superior bioactivity observed in \u003cem\u003ePleurotus florida\u003c/em\u003e compared with \u003cem\u003eP. pulmonarius\u003c/em\u003e suggests a differential expression of secondary metabolite biosynthetic enzymes, such as laccases, peroxidases, and phenol oxidases, which are known to be influenced by environmental and nutritional factors. The role of lignocellulosic substrates, particularly sawdust and corn cob, appears especially important, as they contain lignin derivatives and cellulose fragments that act as metabolic inducers for phenolic and terpenoid biosynthesis. These compounds are well-documented for their ability to disrupt microbial cell wall integrity, alter membrane permeability, and interfere with essential enzymatic processes. From an ecological and biochemical standpoint, substrate composition shapes not only the growth dynamics of \u003cem\u003ePleurotus\u003c/em\u003e species but also the qualitative and quantitative distribution of their secondary metabolites. Lignin-rich materials tend to favor oxidative enzyme activity, which enhances the formation of complex phenolic molecules with antimicrobial and antioxidant properties. In contrast, substrates with lower aromatic content, such as compost waste, may not sufficiently stimulate these enzymatic pathways, leading to a comparatively weaker bioactive profile. These findings support the hypothesis proposed by Diamantopoulou et al. (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) and Kumla et al. (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), who emphasized the substrate-dependent modulation of bioactive compound synthesis in basidiomycetes. The notable antimicrobial spectrum of \u003cem\u003ePleurotus\u003c/em\u003e species, spanning both Gram-positive and Gram-negative bacteria as well as common fungal pathogens, highlights their pharmacological versatility. This broad activity range suggests that the extracts act through multiple molecular mechanisms rather than a single inhibitory pathway. Polyphenolic compounds, for instance, have been shown to chelate metal ions, scavenge reactive oxygen species, and form complexes with microbial proteins, thereby impeding microbial metabolism and replication. The coexistence of flavonoids, alkaloids, and terpenoids may further enhance this effect through synergistic interactions that potentiate cellular disruption and biofilm inhibition. Moreover, the variations observed between the two \u003cem\u003ePleurotus\u003c/em\u003e species could also stem from inherent genetic and enzymatic differences. \u003cem\u003eP. florida\u003c/em\u003e is known for its rapid mycelial colonization and superior enzymatic adaptability, which may enhance its ability to transform complex substrates into high-value secondary metabolites. This aligns with Illuri et al. (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), who attributed the strong antimicrobial capacity of \u003cem\u003eP. florida\u003c/em\u003e to its rich profile of flavonoids, anthraquinones, and terpenoids. Such species-specific attributes underline the importance of genetic and physiological factors in determining the pharmacological potential of cultivated mushrooms. Collectively, these findings reinforce the view that substrate optimization and species selection are central to maximizing the antimicrobial efficacy of \u003cem\u003ePleurotus\u003c/em\u003e mushrooms. By strategically manipulating growth media rich in lignocellulosic components, it may be possible to enhance the yield of bioactive compounds with pharmaceutical relevance. The present results, therefore, contribute to a growing body of evidence supporting the integration of mushroom-based extracts into natural antimicrobial formulations, offering sustainable alternatives to synthetic agents in the ongoing fight against multidrug-resistant pathogens.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study demonstrated that Pleurotus pulmonarius and Pleurotus florida's nutritional quality, phytochemical content, and antibacterial capability are significantly influenced by their substrate composition. Because of its advantageous lignocellulosic composition and aeration qualities, sawdust promoted superior mycelial development and increased antioxidant and antibacterial activity among the studied substrates. The potential of both Pleurotus species as natural sources of bioactive chemicals was confirmed by their significant antibacterial activity against both Gram-positive and Gram-negative bacteria as well as their mild antifungal activities. Only trace amounts of aflatoxins are present, which suggests substrate-related contamination rather than the mushrooms' natural ability to produce poisons. Overall, the results demonstrate the usefulness of agricultural wastes such sawdust and corn cob as effective, secure, and long-lasting substrates for the production of mushrooms. Overall, the findings highlight the value of agro-based wastes such as sawdust and corn cob as efficient, safe, and sustainable substrates for mushroom cultivation. These results emphasize the dual benefits of improved food security and environmental protection through the conversion of agricultural residues into nutritionally rich and bioactive functional foods.\u003c/p\u003e\n\u003ch3\u003eRecommendation\u003c/h3\u003e\n\u003cp\u003eFuture research should focus on optimizing substrate formulations to maximize bioactive compound synthesis while minimizing mycotoxin contamination. The inclusion of nutrient supplements such as agricultural by-products or trace minerals may further enhance yield, antioxidant potency, and overall nutritional quality. Strict sterilization and mycotoxin monitoring protocols are essential to ensure food safety and product consistency. Comparative metabolomic and enzymatic studies are recommended to clarify the biochemical pathways involved in aflatoxin degradation and antioxidant mechanisms in \u003cem\u003ePleurotus\u003c/em\u003e species. Furthermore, scaling up cultivation using locally available agro-wastes such as corn cob, rice straw, and sawdust can promote sustainable mushroom farming, particularly in rural communities. Strengthening collaborations among researchers, policymakers, and farmers will be vital for integrating mushroom cultivation into circular bioeconomy initiatives, advancing environmental conservation, economic resilience, and functional food development.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eCompeting Interests\u003c/h2\u003e\n\u003cp\u003eThe authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e\n\u003ch2\u003eEthics Approval\u003c/h2\u003e\n\u003cp\u003eThis study did not involve human participants or vertebrate animals; therefore, ethics approval was not required. All experimental procedures complied with institutional and national standards for laboratory biosafety and good scientific practice.\u003c/p\u003e\n\u003ch2\u003eConsent to Participate\u003c/h2\u003e\n\u003cp\u003eNot applicable; this study did not involve human participants.\u003c/p\u003e\n\u003ch2\u003eConsent to Publish\u003c/h2\u003e\n\u003cp\u003eNot applicable; this study does not include any person\u0026rsquo;s data, images, or other identifying materials.\u003c/p\u003e\n\u003ch2\u003eFunding\u003c/h2\u003e\n\u003cp\u003eThe authors declare that no funds, grants, or other financial support were received during the preparation and execution of this research work.\u003c/p\u003e\n\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\n\u003cp\u003eAll authors contributed to the study conception and design. Material preparation, data collection, and analysis were performed by Mubarak Muhammed Abdulrazaq, Bajepade Toheeb Taiye, Abdulrahman Muhammad, and Adeyemi Joshua Segun. Mubarak Muhammed Abdulrazaq wrote the first draft of the manuscript, and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003ch2\u003eAcknowledgement\u003c/h2\u003e\n\u003cp\u003eThe authors sincerely acknowledge the Department of Microbiology, Kwara State University, Malete, Nigeria, for providing the laboratory facilities, technical support, and an enabling research environment for this study. Special appreciation is extended to the technical staff of the Microbiology Laboratory for their assistance during the mushroom cultivation and analytical procedures.The authors also appreciate the valuable guidance and constructive suggestions received from colleagues and academic mentors during the design, analysis, and interpretation of data.\u003c/p\u003e\n\u003ch2\u003eData Availability\u003c/h2\u003e\n\u003cp\u003eThe datasets generated and/or analyzed during the current study are available from the corresponding author upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAhmed SEAA, Elbashir AA (2023) Validation of High Performance Liquid Chromatography with Fluorescence Detector Methods for Determination of Aflatoxins in Different Food and Animal Feed Samples. 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Front Sustainable Food Syst 9:1582869\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"world-journal-of-microbiology-and-biotechnology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"wibi","sideBox":"Learn more about [World Journal of Microbiology and Biotechnology](https://www.springer.com/journal/11274)","snPcode":"11274","submissionUrl":"https://submission.nature.com/new-submission/11274/3","title":"World Journal of Microbiology and Biotechnology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Pleurotus species, agro-waste substrates, antioxidant activity, antimicrobial properties, aflatoxin, sustainable cultivation","lastPublishedDoi":"10.21203/rs.3.rs-8055233/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8055233/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAgro-based wastes provide sustainable and nutrient-rich alternatives to synthetic substrates for mushroom cultivation. This study examined the effects of different substrates on the nutritional composition, phytochemical profile, antimicrobial, and antioxidant properties of \u003cem\u003ePleurotus pulmonarius \u003c/em\u003eand \u003cem\u003ePleurotus florida\u003c/em\u003e. Corn cob, sawdust, and compost waste were used to assess their influence on growth performance and bioactive compound production. Standard analytical methods were employed to determine proximate composition, total phenolics, flavonoids, and antioxidant capacity, while antimicrobial activities were evaluated against selected bacterial and fungal pathogens using aqueous extracts. High-performance liquid chromatography (HPLC) was used to quantify aflatoxin residues. Results revealed that sawdust supported the highest mycelial growth and yield, followed by corn cob, while compost waste produced lower performance. \u003cem\u003eP. florida\u003c/em\u003e exhibited superior antioxidant and antibacterial activities compared to \u003cem\u003eP. pulmonarius\u003c/em\u003e, with the highest inhibition recorded against \u003cem\u003eStaphylococcus aureus\u003c/em\u003e and \u003cem\u003eEscherichia coli\u003c/em\u003e. Both species also showed strong antifungal effects against \u003cem\u003eAspergillus\u003c/em\u003especies. Trace concentrations of aflatoxin B₁, B₂, and G₁ detected in the samples suggest possible substrate-related contamination rather than endogenous production. These findings demonstrate that substrate composition significantly affects the nutritional and bioactive properties of \u003cem\u003ePleurotus\u003c/em\u003e species. The use of lignocellulosic agro-wastes such as sawdust and corn cob not only enhances mushroom productivity and bioactivity but also promotes sustainable waste management and functional food development.\u003c/p\u003e","manuscriptTitle":"Influence of Agro-Waste Substrates on the Growth, Nutritional Composition, Phytochemical Profile, Antimicrobial, and Antioxidant Properties of Pleurotus pulmonarius and Pleurotus florida","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-19 15:02:48","doi":"10.21203/rs.3.rs-8055233/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-01-19T15:28:03+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-22T08:02:04+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-16T04:00:36+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"48720090059020871576737667494439563241","date":"2025-12-10T17:41:15+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"38204468106561107664698714847125873709","date":"2025-12-08T15:42:28+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"243988729075737374056839321363634027359","date":"2025-12-08T15:16:38+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"254738554110157943307887113890732381082","date":"2025-11-12T15:15:07+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"311818691929473113049680620174372352970","date":"2025-11-12T03:09:56+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-11-10T11:15:59+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-11-09T17:40:11+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-11-08T13:28:22+00:00","index":"","fulltext":""},{"type":"submitted","content":"World Journal of Microbiology and Biotechnology","date":"2025-11-07T09:01:15+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"world-journal-of-microbiology-and-biotechnology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"wibi","sideBox":"Learn more about [World Journal of Microbiology and Biotechnology](https://www.springer.com/journal/11274)","snPcode":"11274","submissionUrl":"https://submission.nature.com/new-submission/11274/3","title":"World Journal of Microbiology and Biotechnology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"d54e3f87-29e0-4e70-89ed-b57dfa2480cd","owner":[],"postedDate":"November 19th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-05-20T11:38:09+00:00","versionOfRecord":[],"versionCreatedAt":"2025-11-19 15:02:48","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8055233","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8055233","identity":"rs-8055233","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}